Real time electronic cell sensing system and applications for cell-based assays

ABSTRACT

The present invention includes devices, systems, and methods for assaying cells using cell-substrate impedance monitoring. In one aspect, the invention provides cell-substrate impedance monitoring devices that comprise electrode arrays on a nonconducting substrate, in which each of the arrays has an approximately uniform electrode resistance across the entire array. In another aspect, the invention provides cell-substrate monitoring systems comprising one or more cell-substrate monitoring devices comprising multiple wells each having an electrode array, an impedance analyzer, a device station that connects arrays of individual wells to the impedance analyzer, and software for controlling the device station and impedance analyzer. In another aspect, the invention provides cellular assays that use impedance monitoring to detect changes in cell behavior or state. In some preferred aspects, the assays are designed to investigate the affects of compounds on cells, such as cytotoxicity assays. In other preferred aspects, the assays are designed to investigate the compounds that effect IgE-mediated responses of cells to antigens.

This application is divisional of U.S. patent application Ser. No.10/987,732 filed Nov. 12, 2004, now U.S. Pat. No. 7,192,752, entitled“Real Time Electronic Cell Sensing Systems and Applications forCell-based Assays, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/705,447 filed Nov. 10, 2003, now U.S. Pat. No.7,470,533, entitled “Impedance Based Devices and Methods for Use inAssays” which claims priority to U.S. Provisional Application60/397,749, filed Jul. 20, 2002, now expired; U.S. ProvisionalApplication 60/435,400, filed Dec. 20, 2002, now expired; U.S.Provisional Application 60/469,572, filed May 9, 2003, now expired; andPCT application PCT/US03/22557, filed Jul. 18, 2003, now expired. All ofthe applications referred to in this paragraph are incorporated byreference herein.

U.S. patent application Ser. No. 10/987,732, filed Nov. 12, 2004, nowU.S. Pat. No. 7,192,752 is also a continuation-in-part of U.S. patentapplication Ser. No. 10/705,615, entitled “Impedance Based Apparatusesand Methods for Analyzing Cells and Particles”, filed on Nov. 10, 2003,now U.S. Pat. No. 7,459,303, which claims priority to U.S. ProvisionalApplication 60/397,749 filed Jul. 20, 2002, now expired; U.S.Provisional Application 60/435,400, filed Dec. 20, 2002, now expired;U.S. Provisional Application 60/469,572, filed May 9, 2003, now expired;and PCT application PCT/US03/22537, filed Jul. 18, 2003, now expired.All of the applications referred to in this paragraph are incorporatedby reference herein.

U.S. patent application Ser. No. 10/987,732, filed Nov. 12, 2004, nowU.S. Pat. No. 7,192,752 also claims priority to U.S. ProvisionalApplication 60/519,567 filed Nov. 12, 2003, now expired; U.S.Provisional Application 60/542,927 filed Feb. 9, 2004, now expired; andU.S. Provisional Application 60/548,713, filed Feb. 27, 2004, nowexpired. All of the applications referred to in this paragraph areincorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Technical Field

This invention relates to the field of cell-based assays. In particular,the invention provides impedance-based devices, apparatuses and systemsfor analyzing cells and for conducting cell-based assays.

2. Background Art

A. Electronic Analysis of Cells

Bioelectronics is a progressing interdisciplinary research field thatinvolves the integration of biomatereials with electronic devices.Bioelectronic methods have been used for analyzing cells and assayingbiological molecules and cells. In one type of application, cells arecultured on microelectrodes and cell-electrode impedance is measured anddetermined to monitor cellular changes.

In PCT Application No. PCT/US03/22557, entitled “IMPEDANCE BASED DEVICESAND METHODS FOR USE IN ASSAYS”, filed on Jul. 18, 2003, a device fordetecting cells and/or molecules on an electrode surface is disclosed.The device detects cells and/or molecules through measurement ofimpedance changes resulting from the attachment or binding of cellsand/or molecules to the electrode surfaces. A number of embodiments ofthe device is disclosed, together with the apparatuses, system for usingsuch devices to perform certain cell based assays.

B. Allergic Diseases and IgE-Mediated Cell Activation

Allergic diseases, also commonly known as immediate hypersensitivitydisorder are the most common dysfunction of the immune system afflicting20% of all individuals in the United States. The immediatehypersensitivity response can range anywhere from a simple rash or itchyand watery eyes to the most extreme case of anaphylaxis, where theairways are restricted to the point of asphyxiation and death. Due tothe severity of the hypersensitivity responses, the lack of adequatetreatment and the high percentage of the population suffering fromvarious forms of this condition, the pharmaceutical industry has taken akeen interest in developing novel drugs to effectively treat and combatthe symptoms of this disabling and potentially life threateningdisorder.

The primary cells of the immune system that are involved in the allergicresponse are mast cells, basophils and eisonophils. Basophils andeisonophils differentiate in the bone marrow, circulate in the blood andare recruited to the sites of the inflamed tissue in the late-phase ofthe reactions. In contrast, mast cells are normally distributedthroughout the connective tissue and are the involved in the immediatephase of immunoglobulin E (IgE)-mediated allergic reactions (Sharma etal. Clin Rev Allergy Immunol. 2002 April; 22(2):119-48). The initialencounter of an individual with an allergen leads to the production ofIgE, which binds to the high affinity IgE receptor (Fc(epsilon)RI) onthe surface of mast cells causing sensitization of the mast cells.Subsequent encounter with the allergen leads to cross-linking of theFc(epsilon)RI-IgE complex on the surface of mast cells and stimulationof the mast cells to release mediators of immediate hypersensitivity.The cross-linking of receptor-bound IgE on the mast cell surfacetriggers a sequence of intracellular events, collectively referred to asmast cell activation that culminate in the extracellular release ofpotent inflammatory mediators, many of which are stored in the secretorygranules, including histamine. Mast cell activation can be divided intoan interdependent early and late phase. The early phase of mast cellactivation include phosphorylation and activation of protein tyrosinekinases and their substrates, generation of the second messengersinositol-tris phosphate and diacylglycerol, elevation of intracellularcalcium levels and fusion of secretory granules with the membrane(Stassen et al. Crit Rev Immunol. 2002; 22(2):115-40). The late phase ofmast cell activation includes dramatic morphological changes due toremodeling of the actin cytoskeleton, gene expression leading to thesynthesis and secretion of potent inflammatory cytokines and synthesisof lipid mediators that have variety of effects on blood vessels,bronchial smooth muscle and leukocytes.

Based on the various steps involved in the initiation and execution ofthe immediate hypersensitivity response, there are multiple potentialtargets for pharmaceutical intervention. The bulk of the currenttherapies for immediate hypersensitivity disorders such as asthma seekto alleviate the symptoms of the condition rather than directly targetthe underlying cause. However, current promising efforts are underway toneutralize the IgE antibody by administration of humanized monoclonalanti-IgE antibody and to achieve long term alleviation of clinicalsymptoms (D'Amato et al. Monaldi Arch Chest Dis. 2003 January-March;59(1):25-9). Also, the elucidation of the intrinsic signaling pathwaysunderlying IgE-mediated mast cell activation together with the advent ofcombinatorial chemistry provide ample opportunity to employ smallmolecular inhibitors to target key proteins and enzymes involved in mastcell activation. These compounds could potentially provide novelimmunomodulators for the treatment of immediate hypersensitivitydisorder. Small molecular inhibitors of several kinases, including PKCand the tyrosine kinase Syk have provided encouraging preclinicalresults in rodent studies in blocking some immediate hypersensitivityresponses (Seow et al. Eur J Pharmacol. 2002 May 17; 443(1-3):189-96).

An increasing number of companies are utilizing large chemical librariesto screen for potential inhibitors of signaling pathways that maybeinvolved in various disease states. Hence, there is an urgent need forhigh-throughput molecular and cellular assays to screen for potentialmodulators of these signaling pathways. With regards to IgE-mediatedsignaling, the current assays measure mediators that are released intothe media after degranulation. This is accomplished by either measuringthe enzymatic activity of these mediators (Rac and phosphatidylinositol3-kinase regulate the protein kinase B in Fc epsilon RI signaling in RBL2H3 mast cells. J. Immunol. 2001 Feb. 1; 166(3):1627-34), usingradioactive precursors (Guillermot et al. J Cell Sci. 1997 September;110 (Pt 18):2215-25), or by ELISA, quantifying the amount of mediatorsthat are released (Berger et al. Allergy. 2002 July; 57(7):592-9). Theseassays are single point assays or endpoint assays which measure thecumulative release of these mediators and also involve utilization ofreagents and manipulation of the cells, such as fixation or lysis. Thefact that these are single point assays, which utilize expensivereagents such as antibodies and cellular manipulation, does not warrantadaptability for high-throughput analysis that is required to screenlarge chemical libraries.

C. Anticancer Drug Screening and Discovery

In anticancer drug development, the study of the time dependence ofcytotoxic and cell proliferation inhibitory effect of a drug is animportant element for gaining information to use in the development ofclinical dosing strategies. In particular, time dependent IC50's arederived and different time dependent patterns for IC50's are observed(e.g., see Hassan S B, Jonsson E, Larsson R and Karlsson M O in J.Pharmacology and Experimental Therapeutics, 2001, Vol. 299, No. 3, pp1140-1147; Levasseur L M, Slocum H K, Rustum Y M and Greco W R, inCancer Research, 1998, vol. 58, pp 5749-5761). Typically, these studiesused end-point single-measurement assays. Each time point for a doseconcentration of drug or compound applied to the cultured cells requireda separate experiment. This limits the time resolution and the number oftime points of such time-dependent cytotoxicity studies. Thus, newtechnologies or methods that can provide higher time resolution andpermit measurements on many time points are needed.

The present invention further expands the inventions disclosed in PCTApplication No. PCT/US03/22557, entitled “IMPEDANCE BASED DEVICES ANDMETHODS FOR USE IN ASSAYS”, filed on Jul. 18, 2003 and disclosed in U.S.patent application Ser. No. 10/705,447, entitled “IMPEDANCE BASEDDEVICES AND METHODS FOR USE IN ASSAYS,” filed on Nov. 10, 2003. Theinvention provides a real time cell electronic sensing system forconducting cell-based assays based on measurement of cell-substrateimpedance and provides the method for using such a system to performcell-based assays. Furthermore, the present invention is aimed ataddressing the limitations in current methods and technologies forassaying IgE-mediated signaling and cell activation.

SUMMARY OF THE INVENTION

In one aspect, the present invention is directed to a device formonitoring cell-substrate impedance, which device comprises: a) anonconducting substrate; b) two or more electrode arrays fabricated onthe substrate, where each of the two or more electrode arrays comprisestwo electrode structures; and c) at least two connection pads, each ofwhich is located on an edge of the substrate. For each of the two ormore electrode arrays of the device, the first of the two electrodestructures is connected to one of the two or more connection pads, andthe second of the two electrode structures is connected to another ofthe two or more connection pads. Each of the two electrode structures ofthe two or more electrode arrays comprises multiple electrode elements,and each electrode array of the device has an approximately uniformelectrode resistance distribution across the entire array. The substrateof the device has a surface suitable for cell attachment or growth;where cell attachment or growth on said substrate can result in adetectable change in impedance between or among the electrode structureswithin each electrode array. In preferred embodiments, each electrodearray on the substrate of a device of the present invention isassociated with a fluid-impermeable container.

In another aspect, the present invention is directed to a cell-substrateimpedance measurement system comprising: a) at least one multiple-welldevice monitoring cell-substrate impedance, in which at least two of themultiple wells each comprise an electrode array at the bottom of thewell; b) an impedance analyzer; c) a device station capable of engagingthe one or more multiple-well devices and capable of selecting andelectrically connecting electrode arrays within any of the multiplewells in to the impedance analyzer; and d) a software program to controlthe device station and perform data acquisition and data analysis onimpedance values measured by the impedance analyzer. In preferredembodiments of this aspect of the present invention, each electrodearray of the multiple-well device is individually addressed.

In yet another aspect, the present invention provides a method formonitoring cell-substrate impedance using a device of the presentinvention. The method includes: providing a multiple array device of thepresent invention; connecting said multiple array device to an impedanceanalyzer; depositing cells on at least one of the two or more arrays ofthe device; and monitoring cell-substrate impedance on one or morearrays of the device.

In yet another aspect, the present invention provides methods forcalculating a Cell Index for quantifying and comparing cell-substrateimpedance.

In yet another aspect, the present invention provides methods forcalculating resistance of electrical traces connecting an array of acell-substrate monitoring device with a connection pad. Suchcalculations of electrical trace resistance can be used for calculatingCell Index.

In yet another aspect, the present invention provides a method formonitoring cell-substrate impedance using a cell-substrate impedancemeasurement system of the present invention. The method includes:providing a cell-substrate impedance measurement system of the presentinvention, adding cells to at least one well of the multiple-well devicethat comprises an electrode array, and monitoring cell-substrateimpedance from one or more of the wells that comprise cells. Impedancecan be monitored at regular or irregular time intervals. In preferredembodiments, cell-substrate impedance is monitored in at least two wellsof a multiple-well device.

In yet another aspect, the present invention provides a method forperforming real-time cell-based assays investigating the effects of oneor more compound on cells, comprising: providing an above describedcell-substrate impedance measurement system; introducing cells into atleast one well of the system that comprises an electrode array; addingone or more compounds to one or more of the wells containing cells; andmonitoring cell-substrate impedance over the electrode array of the oneor more wells before and after adding the one or more compounds.Preferably, cell-substrate impedance is monitored at regular orirregular time intervals after adding one or more compounds to the oneor more of the wells containing cells. The time dependent impedancechange can provide information about time dependent cell status beforeaddition of the compound and about time dependent cell status under theinteraction of the compound. This information can be used to determinethe effect of a compound on the cells.

In yet another aspect, the present invention provides a method forperforming real-time cytotoxicity assays of at least one compound,comprising: providing an above described cell-substrate impedancemeasurement system; introducing cells into one or more wells of thesystem that comprise an electrode array; adding one or more compounds tothe one or more wells containing cells; and monitoring cell-substrateimpedance of the one or more wells before and after adding the one ormore compounds, wherein the time dependent impedance change providesinformation about time dependent cytotoxicity of the compound orcompounds. Preferably, cell-substrate impedance is monitored at regularor irregular time intervals after adding one or more compounds to theone or more of the wells containing cells. The time dependent impedancechange can provide information about any potential cytotoxic effects ofthe compound.

In one embodiment of the above method, multiple wells with same celltypes are used, wherein different concentrations of a compound are addedto different wells that comprise cells. The method can providetime-dependent and concentration-dependent cytotoxic responses.

In yet another aspect, the present invention provides a method foranalyzing and comparing time-dependent cytotoxic effects of a firstcompound and a second compound on a cell type, comprising: a) performinga real-time cytotoxicity assay on a cell type with the first compoundusing the method described above; b) performing a real-time cytotoxicityassay on said cell type with the second compound using the methoddescribed above; and c) comparing the time-dependent cytotoxic responsesof the first compound and the second compound.

In one embodiment of this method, time-dependent cytotoxic responses aredetermined for a first compound at multiple dose concentrations. Inanother embodiment, time-dependent cytotoxic responses are determinedfor a second compound at multiple dose concentrations. In yet anotherembodiment, time-dependent cytotoxic responses are determined for both afirst compound and a second compound at multiple dose concentrations.

In yet another aspect, the present invention provides methods forcytotoxicity profiling for a compound on multiple cell types,comprising: a) performing real-time cytotoxicity assays on differentcell types with the compound using the method described above, and b)analyzing real-time cytotoxic responses of different cell types to thecompound to provide a cytotoxicity profile of the compound.

In yet another embodiment, the above methods are applied to performcytotoxicity profiling of multiple compounds on multiple cell types.

In yet another aspect, the present invention is directed to method touse electronic impedance technology to assess and quantify themorphological changes that occur in cells as a result of IgE stimulationand IgE receptor cross-linking with an antigen. The method includes:providing a cell-substrate impedance measurement system of the presentinvention; introducing cells into one or more wells of the system,adding IgE to the one or more wells comprising cells; adding an antigento the one or more wells comprising cells and IgE; and monitoringcell-substrate impedance from the one or more wells of the system.Impedance can be monitored before and after adding IgE, and ispreferably measured before adding IgE, after adding IgE and beforeadding antigen, and after adding antigen. The cell-substrate impedancecan be used as an indicator of a cell's response to IgE stimulationthrough cell morphological changes.

In yet another aspect, the present invention is directed at a method toscreen for inhibitors of signaling pathways that are initiated byengagement of the IgE-Fc(epsilon)RI complex by an antigen by utilizingelectronic measurement and sensing of cells. The method includes:providing a cell-substrate impedance measurement system of the presentinvention, introducing cells into: one or more wells of the system,adding at least one test compound to at least one of the one or morewells containing cells; providing at least one control well comprisingcells in the absence of test compound; adding IgE to the one or morewells comprising cells and test compound and to the one or more controlwells; adding an antigen to the one or more wells comprising cells andtest compound and to the one or more control wells; and monitoringcell-substrate impedance from the one or more wells of the system.Impedance can be monitored before and after adding IgE, and ispreferably measured before adding IgE, after adding IgE and beforeadding antigen, and after adding antigen. The cell-substrate impedancecan be used as an indicator of a cell's response to IgE stimulationthrough cell morphological changes. Comparison of the cell-substrateimpedance in one or more wells comprising test compound with one or morecontrol wells provides an assessment of the effect of a test compound onthe cells' response to IgE stimulation. In particular, inhibitors of theIgE response can be identified by their property of reducing thecell-impedance response of cells treated with test compound whencompared with the responses of control cells.

In one embodiment of this aspect, the present invention is directed tomethod to use electronic impedance technology to screen for potentialinhibitors of IgE binding to the high affinity Fc(epsilon)RI receptor onthe surfaces of mast cells or basophils.

In another embodiment of this aspect, the present invention is directedto method of using electronic impedance technology for screening ofsmall molecular inhibitors of key enzymes and proteins involved in thesignaling pathway that ensues from engagement of the high-affinityFc(epsilon)RI receptor of cells in the presence or absence of IgEcross-linking by the antigen.

In yet another aspect, the present invention is directed to method touse electronic impedance technology for target validation purposes ofkey enzymes and proteins involved in the signaling pathway leading fromengagement of the high-affinity FcεRI receptor of cells in the presenceor absence of IgE cross-linking by the antigen. The method includes:providing a cell-substrate impedance measurement system of the presentinvention; introducing genetically modified cells into one or more wellsof the system; providing at least one control well comprising cells thatare not genetically modified; adding IgE to the wells comprisinggenetically modified cells and to the one or more control wells, addingan antigen to the wells comprising genetically modified cells and to theone or more control wells; adding an antigen to the wells comprisinggenetically modified cells and to the one or more control wells;monitoring cell-substrate impedance form the wells comprisinggenetically modified cells and to the one or more control wells; andanalyzing the impedance data to determine the effect of the geneticalteration on the response of cells to IgE stimulation.

In yet another aspect, the present invention is directed to method touse electronic impedance technology for comparing IgE-mediated responsesof cells of different genetic backgrounds. The method can be used forscreening and validating genetic markers such as gene expressionprofiles, gene splicing variants, protein expression profiles, keysingle nucleotide polymorphisms (SNPs) or mutations and other geneticvariants that determine or influence the IgE stimulation, IgE receptorinteractions, antigen-mediated IgE receptor cross-linking, intracellularsignal transduction pathways, and degranulation.

In yet another aspect, the present invention is directed to method touse electronic impedance technology for screening, discovering, andvalidating chemical structures of antigens (allergens) and halfantigens, which lead to IgE cross-linking specifically ornonspecifically.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematic drawings of one design of a cell-substrateimpedance measurement device of the present invention. A) depicts thesubstrate having 16 electrode arrays (or 16 electrode structure units)that are arranged in a 2-row by 8-column configuration on a substrate.B) depicts a single electrode array of a device. C) shows a schematicdrawing of an electrode array, illustrating the requirement ofapproximately uniform distribution of electrode resistance across thearray.

FIG. 2 shows an image of a 16× device of the present invention.

FIG. 3 shows an image of a 96× device of the present invention.

FIG. 4 shows one design of a 16× device station with 6 16× devicesconnected to the station

FIG. 5 shows one design of a 96× device station with a 96-well plateplaced on the station.

FIG. 6 shows different pages from real-time cell electronic sensingsoftware. A) An experimental note page allows the recording of keyinformation about the experiment by the experimenter, such as the goalsand procedures of the experiment. (B) An experimental layout page allowsthe recording of cells, cell number, compound and compound concentrationadded into each well. (C) Test time setting page allows for therecording and control of time intervals used for performingcell-substrate impedance measurement and multiple experimental stepseach having different time interval values and different length timescan be setup. (D) Cell index page is a result page where the softwareautomatically update the measured and derived cell index values for allwells that are under test after the completion of each measurement atpredetermined time interval as setup by the Test time setting page. (E)Experimental data plot page allows for flexible plotting andorganization of experimental data.

FIG. 7 shows real-time monitoring of proliferation of H460 cells seededat different initial cell seeding numbers on a cell substrate monitoringsystem of the present invention. Cell proliferation was continuouslyrecorded every 15 minutes for over 125 hours. The cell growth curves inthe log scale show exponential cell growth or cells in the stationaryphase.

FIG. 8 shows real time monitoring of cell attachment and spreading ofNIH3T3 cells using a cell-substrate impedance monitoring system of thepresent invention. The cells were seeded onto devices coated with eitherpoly-L-lysine or fibronectin. The cell attachment and cell spreadingprocesses on the different coating surfaces were monitored every 3minutes for over 3 hours in real time.

FIG. 9 shows real-time monitoring of morphological changes in Cos-7cells using a cell-substrate impedance monitoring system of the presentinvention. The cells were serum starved for 8 hours and stimulated withor 50 ng/mL EGF. Changes in cell morphology were monitored at 3 minintervals for 2 hours and then 1 hour interval for 14 hours. The initialjump in the signal in EGF-treated cells is due to membrane ruffling andactin dynamics in response to EGF. The arrow indicates the point of EGFstimulation.

FIG. 10 shows a plots of time-dependent cell index for H460 cellstreated by anticancer drug paclitaxel. Different wells of cultured H460cells in their exponential growth phase were treated with differentconcentrations of Paclitaxel. The dynamic response of the cells todifferent doses of paclitaxel was monitored in real time every 15minutes for 50 hours after treatment using a cell-substrate impedancemonitoring system of the present invention.

FIG. 11 shows plots of time-dependent cell index for H460 cells treatedby anticancer drug AC101103. Different wells of cultured H460 cells weretreated at their exponential growth phase with different concentrationsof AC101103. The dynamic response of the cells to different doses ofAC101103 was monitored in real time every 30 minutes for about 20 hoursafter treatment on a cell-substrate impedance monitoring system of thepresent invention.

FIG. 12 shows dynamic drug response curves of A549 cells treated withdoxorubicin. 10,000 A549 cells were seeded in each well of a 16×cell-substrate impedance monitoring device. Cell attachment and cellgrowth were monitored using a cell-substrate impedance monitoring systembefore treatment. When the cells were in exponential growth phase,doxorubicin at different concentration was added to the cells. The timeand drug dose dependent cell response to doxorubicin was recorded inreal time on the as shown in this figure.

FIG. 13 is a plot of cell index recording indicating mast cell responsesto IgE in the presence or absence of antigen. 20,000 RBL-2H3 mast cellswere seeded in each well of a 16× device. Cells were allowed to adhereand grow for 22 hours while being recorded. At 22 hours the cells weretreated with either 1 microgram/mL non-specific mouse IgG or 1microgram/mL anti-DNP mouse IgE. The electronic impedance response ofthe cells were continued to be recorded for an additional 20 hours afterwhich the media in the wells were aspirated and replaced with freshserum free media. The cells were allowed to recover for 30 minutes andthen treated with the 1 microgram/mL of the antigen, DNP-BSA. Theelectronic impedance response of the cells was measured for anadditional 3 hours.

FIG. 14 shows the results of another experiment monitoringcell-electrode impedance responses of RBL-2H3 mast cells sensitized withanti-DNP IgE and activated by the application of DNP-BSA. RBL-2H3 mastcells were seeded at a density of 20,000 cells/well onto the surface ofa 16× device and the impedance was continuously measured and Cell Indexrecorded every 30 minutes using a cell-substrate impedance monitoringsystem of the present invention. Approximately 14 hours after seedingthe cells were incubated with 100 ng/mL anti-DNP IgE followed byapplication of 100 ng/mL DNP-BSA 24 hours later. Impedance measurements(indicated as Cell Index) were performed at 5 minute intervals postDNP-BSA application.

FIG. 15 shows correlation between cell-electrode impedance response ofRBL-2H3 mast cells and cell morphological dynamics and mediator release.(A) RBL-2H3 mast cells were sensitized with 100 ng/mL IgE or treatedwith a control IgG antibody and subsequently activated with DNP-BSA. Thecells were fixed with paraformaldehyde at the indicated time points,permeablized and stained with rhodamine-phalloidin. The cells werevisualized and photographed with a Nikon E-400 immunoflourescencemicroscope equipped with a CCD camera. (B) RBL-2H3 cells were sensitizedwith IgE, activated by the addition of DNP-BSA.

FIG. 16 shows IgE alone-mediated increase in mast cell-electrodeimpedance response and its effect on antigen-mediated activation step.(A) RBL-2H3 cells seeded on microelectronic sensor arrays were leftuntreated or treated with indicated concentration of anti-DNP IgE. Theimpedance value indicated as Cell Index was continuously monitored andrecorded using a cell-substrate impedance monitoring system. (B) RBL-2H3cells that had been sensitized with 15 ng/mL or 1 microgram/mLanti-DNP-IgE were activated by the application of 100 ng/mL DNP-BSA. Theimpedance value indicated as Cell Index was continuously monitored usingthe system.

FIG. 17 shows monitoring of inhibitory effects of Protein kinase Cinhibitor, Bisindolymaleimide on IgE-mediated RBL-2H3 mast cellactivation by cell-electrode impedance measurement. RBL-2H3 mast cellswere sensitized with 100 ng/mL anti-DNP-IgE and then incubated with theindicated concentrations of the Bisindolylmaleimide 1 hour prior toaddition of 100 ng/mL DNP-BSA. The impedance value indicated as CellIndex was monitored using a cell-substrate impedance monitoring systemof the present invention.

FIG. 18 shows the beta-hexosaminidase activity of IgE-mediated RBL-2H3mast cells measured in the presence of pharmacological inhibitors.RBL-2H3 mast cells were sensitized, incubated with DMSO, 16.6 micromolarSU 6656, 5 micromolar U73122, 16.6 micromolar Bisindolylmaleimide, 16.6micromolar Piceatannol and 16.6 micromolar PD 98059 for 1 hour. Thecells were activated by the addition of 100 ng/mL DNP-BSA.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections thatfollow.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entirety. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition that is incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more.”

As used herein, “membrane” is a sheet of material.

As used herein, “biocompatible membrane” means a membrane that does nothave deleterious effects on cells, including the viability, attachment,spreading, motility, growth, or cell division.

When a suspension of viable, unimpaired, epithelial or endothelial cellsis added to a vessel, a surface of the vessel “is suitable for cellattachment” when a significant percentage of the cells are adhering tothe surface of the vessel within twelve hours. Preferably, at least 50%of the cells are adhering to the surface of the vessel within twelvehours. More preferably, a surface that is suitable for cell attachmenthas surface properties so that at least 70% of the cells are adhering tothe surface within twelve hours of plating (i.e., adding cells to thevessel). Even more preferably, the surface properties of a surface thatis suitable for cell attachment results in at least 90% of the cellsadhering to the surface within twelve hours of plating. Most preferably,the surface properties of a surface that is suitable for cell attachmentresults in at least 90% of the cells adhering to the surface withineight, six, four, two hours of plating. To have desired surfaceproperties for cell attachment, the surface may need tochemically-treated (e.g. treatment with an acid and/or with a base),and/or physically treated (e.g. treatment with plasma), and/orbiochemically treated (e.g. coated with one or more molecules orbiomolecules that promotes cell attachment). In the present invention, abiocompatible surface (such as a membrane) preferably is suitable forthe attachment of cells of the type that are to be used in an assay thatuses the biocompatible surface (e.g., membrane), and most preferably,allows the attachment of at least 90% of the cells that contact thebiocompatible surface during the assay.

A “biomolecular coating” is a coating on a surface that comprises amolecule that is a naturally occurring biomolecule or biochemical, or abiochemical derived from or based on one or more naturally occurringbiomolecules or biochemicals. For example, a biomolecular coating cancomprise an extracellular matrix component (e.g., fibronectin,collagens), or a derivative thereof, or can comprise a biochemical suchas polylysine or polyomithine, which are polymeric molecules based onthe naturally occurring biochemicals lysine and ornithine. Polymericmolecules based on naturally occurring biochemicals such as amino acidscan use isomers or enantiomers of the naturally-occurring biochemicals.

An “extracellular matrix component” is a molecule that occurs in theextracellular matrix of an animal. It can be a component of anextracellular matrix from any species and from any tissue type.Nonlimiting examples of extracellular matrix components includelaminins, collagens fibronectins, other glycoproteins, peptides,glycosaminoglycans, proteoglycans, etc. Extracellular matrix componentscan also include growth factors.

An “electrode” is a structure having a high electrical conductivity,that is, an electrical conductivity much higher than the electricalconductivity of the surrounding materials.

As used herein, an “electrode structure” refers to a single electrode,particularly one with a complex structure (as, for example, a spiralelectrode structure), or a collection of at least two electrode elementsthat are electrically connected together. All the electrode elementswithin an “electrode structure” are electrically connected.

As used herein, “electrode element” refers to a single structuralfeature of an electrode structure, such as, for example, a fingerlikeprojection of an interdigitated electrode structure.

As used herein, an “electrode array” or “electrode structure unit” istwo or more electrode structures that are constructed to have dimensionsand spacing such that they can, when connected to a signal source,operate as a unit to generate an electrical field in the region ofspaces around the electrode structures. Preferred electrode structureunits of the present invention can measure impedance changes due to cellattachment to an electrode surface. Non-limiting examples of electrodestructure units are interdigitated electrode structure units andconcentric electrode structure units.

An “electrode bus” is a portion of an electrode that connects individualelectrode elements or substructures. An electrode bus provides a commonconduction path from individual electrode elements or individualelectrode substructures to another electrical connection. In the devicesof the present invention, an electrode bus can contact each electrodeelement of an electrode structure and provide an electrical connectionpath to electrical traces that lead to a connection pad.

“Electrode traces” or “electrically conductive traces” or “electricaltraces”, are electrically conductive paths that extend from electrodesor electrode elements or electrode structures toward one end or boundaryof a device or apparatus for connecting the electrodes or electrodeelements or electrode structures to an impedance analyzer. The end orboundary of a device may correspond to the connection pads on the deviceor apparatus.

A “connection pad” is an area on an apparatus or a device of the presentinvention which is electrically connected to at least one electrode orall electrode elements within at least one electrode structure on anapparatus or a device and which can be operatively connected to externalelectrical circuits (e.g., an impedance measurement circuit or a signalsource). The electrical connection between a connection pad and animpedance measurement circuit or a signal source can be direct orindirect, through any appropriate electrical conduction means such asleads or wires. Such electrical conduction means may also go throughelectrode or electrical conduction paths located on other regions of theapparatus or device.

“Interdigitated” means having projections coming one direction thatinterlace with projections coming from a different direction in themanner of the fingers of folded hands (with the caveat thatinterdigitated electrode elements preferably do not contact oneanother).

As used herein, a “high probability of contacting an electrode element”means that, if a cell is randomly positioned within the sensor area of adevice or apparatus of the present invention, the probability of a cell(or particle) contacting on an electrode element, calculated from theaverage diameter of a cell used on or in a device or apparatus of thepresent invention, the sizes of the electrode elements, and the size ofthe gaps between electrode elements, is greater than about 50%, morepreferably greater than about 60%, yet more preferably greater thanabout 70%, and even more preferably greater than about 80%, greater thanabout 90%, or greater than about 95%.

As used herein, “at least two electrodes fabricated on said substrate”means that the at least two electrodes are fabricated or made orproduced on the substrate. The at least two electrodes can be on thesame side of the substrate or on the different side of the substrate.The substrate may have multiple layers, the at least two electrodes canbe either on the same or on the different layers of the substrate.

As used herein, “at least two electrodes fabricated to a same side ofsaid substrate” means that the at least two electrodes are fabricated onthe same side of the substrate.

As used herein, “at least two electrodes fabricated to a same plane ofsaid substrate” means that, if the nonconducting substrate has multiplelayers, the at least two electrodes are fabricated to the same layer ofthe substrate.

As used herein, “said . . . electrodes [or electrode structures] havesubstantially the same surface area” means that the surface areas of theelectrodes referred to are not substantially different from each other,so that the impedance change due to cell attachment or growth on any oneof the electrodes (or electrode structures) referred to will contributeto the overall detectable change in impedance to a same or similardegree as the impedance change due to cell attachment or growth on anyother of the electrodes (or electrode structures) referred to. In otherwords, where electrodes (or electrode structures) have substantially thesame surface area, any one of the electrodes can contribute to overallchange in impedance upon cell attachment or growth on the electrode. Inmost cases, the ratio of surface area between the largest electrode andthe smallest electrode that have “substantially the same surface area”is less than 10. Preferably, the ratio of surface area between thelargest electrode and the smallest electrode of an electrode array isless than 5, 4, 3, 2, 1.5, 1.2 or 1.1. More preferably, the at least twoelectrodes of an electrode structure have nearly identical or identicalsurface area.

As used herein, “said device has a surface suitable for cell attachmentor growth” means that the electrode and/or non-electrode area of theapparatus has appropriate physical, chemical or biological propertiessuch that cells of interest can viably attach on the surface and newcells can continue to attach, while the cell culture grows, on thesurface of the apparatus. However, it is not necessary that the device,or the surface thereof, contain substances necessary for cell viabilityor growth. These necessary substances, e.g., nutrients or growthfactors, can be supplied in a medium. Preferably, when a suspension ofviable, unimpaired, epithelial or endothelial cells is added to the“surface suitable for cell attachment” when at least 50% of the cellsare adhering to the surface within twelve hours. More preferably, asurface that is suitable for cell attachment has surface properties sothat at least 70% of the cells are adhering to the surface within twelvehours of plating (i.e., adding cells to the chamber or well thatcomprises the said device). Even more preferably, the surface propertiesof a surface that is suitable for cell attachment results in at least90% of the cells adhering to the surface within twelve hours of plating.Most preferably, the surface properties of a surface that is suitablefor cell attachment results in at least 90% of the cells adhering to thesurface within eight, six, four, two hours of plating.

As used herein, “detectable change in impedance between or among saidelectrodes” (or “detectable change in impedance between or among saidelectrode structures”) means that the impedance between or among saidelectrodes (or electrode structures) would have a significant changethat can be detected by an impedance analyzer or impedance measurementcircuit when molecule binding reaction occurs on the electrode surfaces.The impedance change refers to the difference in impedance values whenmolecule binding reaction occurs on the electrode surface of theapparatus and when no molecular reaction occurs on the electrodesurface. Alternatively, the impedance change refers to the difference inimpedance values when cells are attached to the electrode surface andwhen cells are not attached to the electrode surface, or when thenumber, type, activity, adhesiveness, or morphology of cells attached tothe electrode-comprising surface of the apparatus changes. In mostcases, the change in impedance is larger than 0.1% to be detectable.Preferably, the detectable change in impedance is larger than 1%, 2%,5%, or 8%. More preferably, the detectable change in impedance is largerthan 10%. Impedance between or among electrodes is typically a functionof the frequency of the applied electric field for measurement.“Detectable change in impedance between or among said electrodes” doesnot require the impedance change at all frequencies being detectable.“Detectable change in impedance between or among said electrodes” onlyrequires a detectable change in impedance at any single frequency (ormultiple frequencies). In addition, impedance has two components,resistance and reactance (reactance can be divided into two categories,capacitive reactance and inductive reactance). “Detectable change inimpedance between or among said electrodes” requires only that eitherone of resistance and reactance has a detectable change at any singlefrequency or multiple frequencies. In the present application, impedanceis the electrical or electronic impedance. The method for themeasurement of such impedance is achieved by, (1) applying a voltagebetween or among said electrodes at a given frequency (or multiplefrequencies, or having specific voltage waveform) and monitoring theelectrical current through said electrodes at the frequency (or multiplefrequencies, or having specific waveform), dividing the voltageamplitude value by the current amplitude value to derive the impedancevalue; (2) applying an electric current of a single frequency component(or multiple frequencies or having specific current wave form) throughsaid electrodes and monitoring the voltage resulted between or amongsaid electrodes at the frequency (or multiple frequencies, or havingspecific waveform), dividing the voltage amplitude value by the currentamplitude value to derive the impedance value; (3) other methods thatcan measure or determine electric impedance. Note that in thedescription above of “dividing the voltage amplitude value by thecurrent amplitude value to derive the impedance value”, the “division”is done for the values of current amplitude and voltage amplitude atsame frequencies. Measurement of such electric impedance is anelectronic or electrical process that does not involve the use of anyreagents.

As used herein, “said at least two electrodes have substantiallydifferent surface area” means that the surface areas of any electrodesare not similar to each other so that the impedance change due to cellattachment or growth on the larger electrode will not contribute to theoverall detectable impedance to a same or similar degree as theimpedance change due to cell attachment or growth on the smallerelectrodes. Preferably, any impedance change due to cell attachment orgrowth on the larger electrode is significantly smaller than theimpedance change due to cell attachment or growth on the smallerelectrode. Ordinarily, the ratio of surface area between the largestelectrode and the smallest electrode is more than 10. Preferably, theratio of surface area between the largest electrode and the smallestelectrode is more than 20, 30, 40, 50 or 100.

As used herein, “multiple pairs of electrodes or electrode structuresspatially arranged according to wells of a multi-well microplate” meansthat the multiple pairs of electrodes or electrode structures of adevice or apparatus are spatially arranged to match the spatialconfiguration of wells of a multi-well microplate so that, whendesirable, the device can be inserted into, joined with, or attached toa multiwell plate (for example, a bottomless multiwell plate) such thatmultiple wells of the multi-well microplate will comprise electrodes orelectrode structures.

As used herein, “arranged in a row-column configuration” means that, interms of electric connection, the position of an electrode, an electrodearray or a switching circuit is identified by both a row position numberand a column position number.

As used herein, “each well contains substantially same number . . . ofcells” means that the lowest number of cells in a well is at least 50%of the highest number of cells in a well. Preferably, the lowest numberof cells in a well is at least 60%, 70%, 80%, 90%, 95% or 99% of thehighest number of cells in a well. More preferably, each well containsan identical number of cells.

As used herein, “each well contains . . . same type of cells” meansthat, for the intended purpose, each well contains same type of cells;it is not necessary that each well contains exactly identical type ofcells. For example, if the intended purpose is that each well containsmammalian cells, it is permissible if each well contains same type ofmammalian cells, e.g., human cells, or different mammalian cells, e.g.,human cells as well as other non-human mammalian cells such as mice,goat or monkey cells, etc.

As used herein, “each well contains . . . serially differentconcentration of a test compound” means that each well contains a testcompound with a serially diluted concentrations, e.g., an one-tenthserially diluted concentrations of 1 M, 0.1 M, 0.01 M, etc.

As used herein, “dose-response curve” means the dependent relationshipof response of cells on the dose concentration of a test compound. Theresponse of cells can be measured by many different parameters. Forexample, a test compound is suspected to have cytotoxicity and causecell death. Then the response of cells can be measured by percentage ofnon-viable (or viable) cells after the cells are treated by the testcompound.

As used herein, “the electrodes have, along the length of themicrochannel, a length that is substantially less than the largestsingle-dimension of a particle to be analyzed” means that the electrodeshave, along the length of the microchannel, a length that is at leastless than 90% of the largest single-dimension of a particle to beanalyzed. Preferably, the electrodes have, along the length of themicrochannel, a length that is at least less than 80%, 70%, 60%, 50%,40%, 30%, 20%, 10%, 5% of the largest single-dimension of a particle tobe analyzed.

As used herein, “the microelectrodes span the entire height of themicrochannel” means that the microelectrodes span at least 70% of theentire height of the microchannel. Preferably, microelectrodes span atleast 80%, 90%, 95% of the entire height of the microchannel. Morepreferably, microelectrodes span at least 100% of the entire height ofthe microchannel.

As used herein, “an aperture having a pore size that equals to or isslightly larger than size of said particle” means that aperture has apore size that at least equals to the particle size but less than 300%of the particle size. Here both pore size and particle size are measuredin terms of single dimension value.

As used herein, “microelectrode strip or electrode strip” means that anon-conducting substrate strip on which electrodes or electrodestructure units are fabricated or incorporated. The non-limitingexamples of the non-conducting substrate strips include polymermembrane, glass, plastic sheets, ceramics, insulator-on-semiconductor,fiber glass (like those for manufacturing printed-circuits-board).Electrode structure units having different geometries can be fabricatedor made on the substrate strip by any suitable microfabrication,micromachining, or other methods. Non-limiting examples of electrodegeometries include interdigitated electrodes, circle-on-line electrodes,diamond-on-line electrodes, castellated electrodes, or sinusoidalelectrodes. Characteristic dimensions of these electrode geometries mayvary from as small as less than 5 micron, or less than 10 micron, to aslarge as over 200 micron, over 500 micron, over 1 mm. The characteristicdimensions of the electrode geometries refer to the smallest width ofthe electrode elements, or smallest gaps between the adjacent electrodeelements, or size of a repeating feature on the electrode geometries.The microelectrode strip can be of any geometry for the presentinvention. One exemplary geometry for the microelectrode strips isrectangular shape—having the width of the strip between less than 50micron to over 10 mm, and having the length of the strip between lessthan 60 micron to over 15 mm. An exemplary geometry of themicroelectrode strips may have a geometry having a width of 200 micronand a length of 20 mm. A single microelectrode strip may have twoelectrodes serving as a measurement unit, or multiple suchtwo-electrodes serving as multiple measurement units, or a singleelectrode structure unit as a measurement unit, or multiple electrodestructure units serving as multiple electrode structure units. In oneexemplary embodiment, when multiple electrode structure units arefabricated on a single microelectrode strip, these electrode structureunits are positioned along the length direction of the strip. Theelectrode structure units may be of squared-shape, or rectangular-shape,or circle shapes. Each of electrode structure units may occupy size fromless than 50 micron by 50 micron, to larger than 2 mm×2 mm.

As used herein, “sample” refers to anything which may contain a moietyto be isolated, manipulated, measured, quantified, detected or analyzedusing apparatuses, microplates or methods in the present application.The sample may be a biological sample, such as a biological fluid or abiological tissue. Examples of biological fluids include suspension ofcells in a medium such as cell culture medium, urine, blood, plasma,serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears,mucus, amniotic fluid or the like. Biological tissues are aggregates ofcells, usually of a particular kind together with their intercellularsubstance that form one of the structural materials of a human, animal,plant, bacterial, fungal or viral structure, including connective,epithelium, muscle and nerve tissues. Examples of biological tissuesalso include organs, tumors, lymph nodes, arteries and individualcell(s). The biological samples may further include cell suspensions,solutions containing biological molecules (e.g. proteins, enzymes,nucleic acids, carbonhydrates, chemical molecules binding to biologicalmolecules).

As used herein, a “liquid (fluid) sample” refers to a sample thatnaturally exists as a liquid or fluid, e.g., a biological fluid. A“liquid sample” also refers to a sample that naturally exists in anon-liquid status, e.g., solid or gas, but is prepared as a liquid,fluid, solution or suspension containing the solid or gas samplematerial. For example, a liquid sample can encompass a liquid, fluid,solution or suspension containing a biological tissue.

B. Devices and Systems for Monitoring Cell-Substrate Impedance

Devices for Measuring Cell-Substrate Impedance

The present invention includes devices for measuring cell-substrateimpedance that comprise a nonconducting substrate; two or more electrodearrays fabricated on the substrate, where each of the two or moreelectrode arrays comprises two electrode structures; and at least twoconnection pads, each of which is located on an edge of the substrate.Each electrode array of the device has approximately uniform electroderesistance across the entire array. The substrate of the device has asurface suitable for cell attachment or growth; where cell attachment orgrowth on said substrate can result in a detectable change in impedancebetween or among the electrode structures within each electrode array.

An electrode array is two or more electrode structures that areconstructed to have dimensions and spacing such that they can, whenconnected to a signal source, operate as a unit to generate anelectrical field in the region of spaces around the electrodestructures. An electrode structure refers to a single electrode,particularly one with a complex structure. (For example, an electrodestructure can comprise two or more electrode elements that areelectrically connected together.) In devices of the present invention,an electrode array comprises two electrode structures, each of whichcomprises multiple electrode elements, or substructures. In preferredembodiments of the present invention, the electrode structures of eachof the two or more electrode arrays of a device have substantially thesame surface area. In preferred embodiments of a device of the presentinvention, each of the two or more electrode arrays of a device comprisetwo electrode structures, and each electrode structure comprisesmultiple electrode elements. Each of the two electrode structures of anelectrode array is connected to a separate connection pad that islocated at the edge of the substrate.

Thus, in devices of the present invention, for each of the two or moreelectrode arrays of the device, the first of the two electrodestructures is connected to one of the two or more connection pads, andthe second of the two electrode structures is connected to another ofthe two or more connection pads. Preferably, each array of a device isindividually addressed, meaning that the electrical traces andconnection pads of the arrays are configured such that an array can beconnected to an impedance analyzer in such a way that a measuringvoltage can be applied across a single array at a given time by usingswitches (such as electronic switches).

Each electrode array of the device has an approximately uniformelectrode resistance distribution across the entire array. By “uniformresistance distribution across the array” is meant that when ameasurement voltage is applied across the electrode structures of thearray, the electrode resistance at any given location of the array isapproximately equal to the electrode resistance at any other location onthe array. Preferably, the electrode resistance at a first location onan array of the device and the electrode resistance at a second locationon the same array does not differ by more than 30%. More preferably, theelectrode resistance at a first location on an array of the device andthe electrode resistance at a second location on the same array does notdiffer by more than 15%. Even more preferably, the electrode resistanceat a first location on an array of the device and a second location onthe same array does not differ by more than 5%. More preferably yet, theelectrode resistance at a first location on an array of the device and asecond location on the same array does not differ by more than 2%.

For a device of the present invention, preferred arrangements for theelectrode elements, gaps between the electrodes and electrode buses in agiven electrode array are used to allow all cells, no matter where theyland and attach to the electrode surfaces, to contribute similarly tothe total impedance change measured for the electrode array. Thus, it isdesirable to have similar electric field strengths at any two locationswithin any given array of the device when a measurement voltage isapplied to the electrode array. At any given location of the array, thefield strength is related to the potential difference between thenearest point on a first electrode structure of the array and thenearest point on a second electrode structure of the array. It istherefore desirable to have similar electric potential drops across theelectrode elements and across the electrode buses of a given array.Based on this requirement, it is preferred to have an approximatelyuniform electrode resistance distribution across the whole array wherethe electrode resistance at a location of interest is equal to the sumof the electrode resistance between the nearest point on a firstelectrode structure (that is the point on the first electrode structurenearest the location of interest) and a first connection pad connectedto the first electrode structure and the electrode resistance betweenthe nearest point on a second electrode structure (that is the point onthe first electrode structure nearest the location of interest) and asecond connection pad connected to the second electrode structure.

Devices of the present invention are designed such that the arrays ofthe device have an approximately uniform distribution across the wholearray. This can be achieved, for example, by having electrode structuresand electrode buses of particular spacing and dimensions (lengths,widths, thicknesses and geometrical shapes) such that the resistance atany single location on the array is approximately equal to theresistance at any single other location on the array. In mostembodiments, the electrode elements (or electrode structures) of a givenarray will have even spacing and be of similar thicknesses and widths,the electrode buses of a given array will be of similar thicknesses andwidths, and the electrode traces leading from a given array to aconnection pad will be of closely similar thicknesses and widths. Thus,in these preferred embodiments, an array is designed such that thelengths and geometrical shapes of electrode elements or structures, thelengths and geometrical shapes of electrode traces, and the lengths andgeometrical shapes of buses allow for approximately uniform electroderesistance distribution across the array.

In some preferred embodiments of cell-substrate impedance measurementdevices, electrode structures comprise multiple electrode elements, andeach electrode element connects directly to an electrode bus. Electrodeelements of a first electrode structure connect to a first electrodebus, and electrode elements of a second electrode structure connect to asecond electrode bus. In these embodiments, each of the two electrodebuses connects to a separate connection pad via an electrical trace.Although the resistances of the traces contribute to the resistance at alocation on the array, for any two locations on the array the traceconnections from the first bus to a first connection pad and from thesecond bus to a second connection pad are identical. Thus, in thesepreferred embodiments trace resistances do not need to be taken intoaccount in designing the geometry of the array to provide for uniformresistances across the array.

Taking the electrode array in FIG. 1C as an example, it is desirablethat cells attached at location B and cells attached at location Acontribute similarly to the total impedance change. Thus, it isdesirable to have similar electric field strengths at location A andlocation B when a measurement voltage is applied to the electrode array.It is thus desirable to have similar electric potential drops across theelectrode elements and across the electrode buses. Based on thisrequirement, it is preferred to have an approximately uniform electroderesistance distribution across the whole array where the electroderesistance at a location of interest is equal to the sum of theelectrode resistances between the electrode points at the location andthe two connection pads connected to the electrode array. The electroderesistance at location A and location B in FIG. 1C is given byR _(location) _(—) _(A) =R _(trace1) +R _(1-to-A1) +R _(A1-to-A) +R_(A-to-A2) +R _(A2-to-2) +R _(trace2)  (1)R _(location) _(—) _(B) =R _(trace1) +R _(1-to-B1) +R _(B1-to-B) +R_(B-to-B2) +R _(B2-to-2) +R _(trace2)  (2)where R_(trace1) is the impedance (i.e., resistance, since the reactioncomponent is very very small) of the electrical connection trace from aconnection pad to location 1 on the top electrode bus; R_(trace2) is theimpedance (i.e., resistance, since the reaction component is very verysmall) of the electrical connection trace from a connection pad tolocation 2 on the bottom electrode bus; R_(1-to-A1) is the impedance(i.e., resistance, the reaction component is very very small) betweenlocation 1 and location A1 along the top electrode bus; R_(2-to-A2) isthe impedance (i.e., resistance, the reaction component is very verysmall) between location 2 and location A2 along the bottom electrodebus; R_(A1-to-A) is the impedance (i.e., resistance, the reactioncomponent is very very small) between location A1 on the top electrodebus and location A along a connected electrode element; R_(A2-to-A) isthe impedance (i.e., resistance, the reaction component is very verysmall) between location A2 on the bottom electrode bus and location Aalong another connected electrode element. The requirement for anapproximately uniform electrode resistance is that R_(location) _(—)_(A) is similar to R_(location) _(—) _(B).

Electrode structures shown in FIG. 1C are designed to satisfy therequirement of approximately uniform electrode resistance. In FIG. 1C,the electrodes or electrode elements are circle-on-a-line configurationwith diameter of the circle being about 90 microns, the line width about30 microns and the gaps between lines about 80 microns. The innerdiameter of the electrode bus is about 5.75 mm and the outer diameterfor the electrode bus is about 6.070 mm.

In preferred embodiments of the present invention, for the measurementof cell-substrate impedance, an impedance analyzer is connectedindirectly through electronic switches to the connection pads on thesubstrate. The measured impedance Z_(total) at the impedance analyzer isgiven byZ _(total) =Z _(switch) +Z _(trace) +Z _(electrode-array)  (3)where Z_(switch) is the impedance of electronic switch at its on stage,Z_(trace) is the impedance of the electrical connection traces on thesubstrate between the connection pads and the electrode buses,Z_(electrode-array) is the impedance of the electrode array with orwithout cell-being present. Major components of both Z_(switch) andZ_(trace) are electric resistance. The contribution of Z_(switch) andZ_(trace) to total measured impedance Z_(total) can be removed from themeasured impedance if these values can be determined. By choosingelectronic switches of good quality, the Z_(switch) values can benearly-constant for different switches. Thus, the contribution ofZ_(switch) to total measured impedance Z_(total) can be removed from themeasured impedance. Z_(trace) is somewhat difficult to remove becausedifferent electrode arrays may have different impedance values and alsobecause there is also a thickness variation in the conductive thin filmused for making the electrode structures between different devices. Foraccurate measurement of cell-substrate impedance, it is preferred tohave small electrical connection trace (or electrical traces) impedanceZ_(trace). For this reason, it is desirable to have electrical traces oflarge width, which will lead to a reduced size for the electrode arrayarea. For this reason, in one embodiment of the device of thepresentation, in order to have reasonably wide electrical traces, thediameter of the electrode array area, as exemplified in FIG. 1B as theinner diameter of arc-shaped electrode buses, was made smaller than thebottom well diameter of a standard microtiter plate. To ensure theelectrode buses are not exposed to solution during the assay, a platehaving wells whose with a bottom diameter is small enough to exclude theelectrode buses from the interior of the wells can be attached to thesubstrate.

In preferred embodiments of the present invention, a device formonitoring cell-substrate impedance has two or more electrode arraysthat share a connection pad. Preferably one of the electrode structuresof at least one of the electrode arrays of the device is connected to aconnection pad that also connects to an electrode structure of at leastone other of the electrode arrays of the device. Preferably for at leasttwo arrays of the device, each of the two or more arrays has a firstelectrode structure connected to a connection pad that connects with anelectrode structure of at least one other electrode array, and each ofthe two or more arrays has a second electrode structure that connects toa connection pad that does not connect with any other electrodestructures or arrays of the device. Thus, in preferred designs of adevice there are at least two electrode arrays each of which has a firstelectrode structure that is connected to a common connection pad and asecond electrode structure that is connected to an independentconnection pad.

In some preferred embodiments of the present invention, each of theelectrode structures of an array is connected to an electrode bus thatis connected to one of the two or more connection pads of the device viaan electrically conductive trace. In preferred embodiments, each of thetwo electrode structures is connected to a single bus, such that eacharray connects to two buses, one for each electrode structures. In thisarrangement, each of the two buses connects to a separate connection padof the substrate.

The electrically conductive traces that connect a bus with a connectioncan be fabricated of any electrically conductive material. The tracescan be localized to the surface of the substrate, and can be optionallycovered with an insulating layer. Alternatively the traces can bedisposed in a second plane of the substrate. Description of arrangementsand design of electrically conductive traces on impedance measurementdevices can be found in parent U.S. patent application Ser. No.10/705,447, herein incorporated by reference for disclosure onfabrication and design of electrically conductive trace on substrates.

FIG. 1A shows an example of the device of the present invention. Thisdevice comprises a glass substrate (101) shown with 16 electrode arraysfabricated on the substrate. Each electrode array (102) comprises twoelectrode structures (shown in detail in FIG. 1B). Each electrode arrayconnects to two electrical traces (103), with each of the two tracesconnected one of the two electrode structures. These electricalconnection traces (103) from the electrode array (102) are connected tothe connection pads (104) at the edges of the substrate (101). As shownin FIG. 1A, each the four electrode arrays in each of four quarters onthe substrate (101) have one of their electrical connection traces (103)connected to a common connection pad (104). Thus, for the entire devicethere are four common connection pads (104), one for each quarter of thedevice. In addition, each electrode array has a separate electricalconnection trace (103), connecting to an independent connection pad(104). Thus, there are total 20 connection pads (104) at the edges ofthe substrate (101).

An example of an electrode array that can be used on a device of thepresent invention (such as that of FIG. 1A) is depicted in FIG. 1B.Here, a single electrode array is shown. The electrode array has twoelectrode structures, where each electrode structure comprises multipleelectrode elements (105) shown here having a circle-on-line geometry. Inthis electrode array structure, electrode elements (105) of oneelectrode structure of the array alternate with electrode elements (105)of the other electrode structure of the array. Each of the electrodestructures is independently connected to its electrode bus (106), inthis case, by means of direct connection of the electrode elements (105)to the electrode bus (106). Each electrode bus (106) forms an arc aroundthe perimeter of the array, where the two buses of the array do not abutor overlap. The electrically conductive connection traces (103 in FIG.1A) connect each bus with a connection pad (104 in FIG. 1A) on the edgeof the substrate (101 in FIG. 1A)

Appropriate electronic connection means such as metal clips engaged ontothe connection pads on the substrate and connectedprinted-circuit-boards can be used for leading the electronicconnections from the connection pads on the devices to externalelectronic circuitry (e.g. an impedance analyzer). Description of thedesign of cell-substrate impedance devices and their manufacture can befound in U.S. patent application Ser. No. 10/705,447, hereinincorporated by reference for description and disclosure of the design,features, and manufacture of impedance device comprising electrodearrays.

Preferably the nonconducting substrate is planar, and is flat orapproximately flat. Exemplary substrates can comprise many materials,including, but not limited to, silicon dioxide on silicon,silicon-on-insulator (SOI) wafer, glass (e.g., quartz glass, lead glassor borosilicate glass), sapphire, ceramics, polymer, fiber glass,plastics, e.g., polyimide (e.g. Kapton, polyimide film supplied byDuPont), polystyrene, polycarbonate, polyvinyl chloride, polyester,polypropylene and urea resin. Preferably, the substrate and the surfaceof the substrate are not going to interfere with molecular bindingreactions that will occur at the substrate surface. For cell-substrateimpedance monitoring, any surface of the nonconducting substrate thatcan be exposed to cells during the use of a device of the presentinvention is preferably biocompatible. Substrate materials that are notbiocompatible can be made biocompatible by coating with anothermaterial, such as polymer or biomolecular coating.

All or a portion of the surface of a substrate can be chemicallytreated, including but not limited to, modifying the surface such as byaddition of functional groups, or addition of charged or hydrophobicgroups.

Descriptions of electrode arrays used for impedance measurement thatapply to the devices of the present invention are described in parentU.S. patent application Ser. No. 10/705,447, herein incorporated byreference for all disclosure relating to electrode arrays (or structuralunits), electrode structures, electrode materials, electrode dimensions,and methods of manufacturing electrodes on substrates.

Preferred electrode arrays for devices of the present invention includearrays comprising two electrode structures, such as, for example, spiralelectrode arrays and interdigitated arrays. In some preferred devices ofthe present invention, electrode arrays are fabricated on a substrate,in which the arrays comprises two electrode structures, each of whichcomprises multiple circle-on-line electrode elements, in which theelectrode elements of one structure alternate with the electrodeelements of the opposite electrode structure. For example, FIG. 1Bdepicts such an array.

Preferably, the electrode elements (or electrode structures) of an arrayof the present device of the present invention are of approximatelyequal widths. Preferably the electrode elements (or electrodestructures) of an array of the present device of the present inventionare greater than 30 microns in width, more preferably from about 50 toabout 300 microns in width, and more preferably yet about 90 microns inwidth.

Preferably, the electrode elements (or electrode structures) of an arrayof the present device of the present invention are approximately evenlyspaced. Preferably, the gap between electrode elements (or electrodestructures) of an array of the present device of the present inventionis less than 50 microns in width, more preferably from about 5 to about30 microns in width, and more preferably yet about 20 microns in width.

A device of the present invention can include one or morefluid-impermeable receptacles which serve as fluid containers. Suchreceptacles may be reversibly or irreversibly attached to or formedwithin the substrate or portions thereof (such as, for example, wellsformed as in a microtiter plate). In another example, the device of thepresent invention includes microelectrode strips reversibly orirreversibly attached to plastic housings that have openings thatcorrespond to electrode structure units located on the microelectrodestrips. Suitable fluid container materials comprise plastics, glass, orplastic coated materials such as ceramics, glass, metal, etc.Descriptions and disclosure of devices that comprise fluid containerscan be found in parent U.S. patent application Ser. No. 10/705,447,herein incorporated by reference for all disclosure of fluid containersand fluid container structures that can engage a substrate comprisingelectrodes for impedance measurements, including their dimensions,design, composition, and methods of manufacture.

In preferred embodiments, each electrode array on the substrate of adevice of the present invention is associated with a fluid-impermeablecontainer or receptacle. Preferably, the device of the present inventionis assembled to a bottomless, multiwell plastic plate or strip with afluid tight seal, as shown, for example, in FIG. 2 and FIG. 3. Thedevice is assembled such that a single array of the substrate is at thebottom of a receptacle or well. Preferably, each array of a device isassociated with a well of a multiwell plate. In some preferredembodiments, a multiwell device for cell-substrate impedance measurementhas “non-array” wells that are attached to the substrate but notassociated with arrays. Such wells can optionally be used for performingnon-impedance based assays, or for viewing cells microscopically.

The design and assembly of multiwell impedance measurement devices isdescribed in parent U.S. patent application Ser. No. 10/705,447, hereinincorporated by reference for disclosure of multiwell impedancemeasurement devices, including their design, composition, andmanufacture. A device of the present invention preferably has between 2and 1,536 wells, more preferably between 4 and 384 wells, and even morepreferably, between 16 and 96 wells, all or less than all or which areassociated with electrode arrays.

In some preferred embodiments, commercial tissue culture plates can beadapted to fit a device of the present invention. Bottomless plates mayalso be custom-made to preferred dimensions. Preferably, well diametersare from about 1 millimeter to about 20 millimeters, more preferablyfrom about 2 millimeters to about 8 millimeters at the bottom of thewell (the end disposed on the substrate). The wells can have a uniformdiameter or can taper toward the bottom so that the diameter of thecontainer at the end in contact with the substrate is smaller than thediameter of the opposing end.

Preferred Devices

The following descriptions of devices are not intended to limit theinvention in any way.

FIG. 2 is an image of one design of a 16× device with a glass substrate(201) having 16 microfabricated electrode arrays mounted to aprinted-circuit-board (208) via metal clips (209). The metal clips (209)are engaged onto connection pads (204) on the substrate and are solderedto the connection lines (210) on the printed-circuit-board (208). Theconnection lines (210) on the printed-circuit-board (208) in turn areconnected to the connection pins (211) located on the edge of thedevice. A bottomless plastic 16 well strip (207) is bonded to thesubstrate (201) via a double-sided pressure sensitive adhesive, forming16 individual fluid containers. In this device, the bottom diameter ofthe fluid containers is about 5 mm.

FIG. 3 shows an image of one design of a 96× device with six glasssubstrates each having either 15 or 16 microfabricated electrode arrays.FIG. 3A shows the bottom side of the device with open wells facingdownwards and FIG. 3B shows the device with a plastic lid with wellsopen well facing upwards. The six substrates (301) are mounted andsealed to a bottomless 96 well plate (307) so that 95 of 96 wellscomprise an electrode array on the bottom surface of the wells (when theopen wells face upwards). One of 96 wells does not have electrodes (asindicated (312) in FIG. 3A). The well bottom diameter is about 5 mm.Metal clips are engaged onto the connection pads on the substrate andare soldered to the connection lines on the top side (when the 96-wellplate is placed with open wells facing upward, as shown in FIG. 3B) ofsmall printed-circuit-boards (308) located on the ends of the substrates(301). The printed-circuit-boards (308) are double-sided, and samenumber of connection lines exists on both sides of the boards atpositions opposite to each other. The connection lines on the top sideof the board are connected through conductive vias to connection lineson the bottom side, where the electronic connections to a device stationare made.

Methods of Use

The present invention also includes methods of using a device of thepresent invention that comprises fluid containers situated overelectrode arrays to measure cell-substrate impedance. Such methodsinclude: providing a device of the present invention that comprisesfluid containers situated over electrode arrays, attaching an impedanceanalyzer to a device of the present invention, adding cells to one ormore fluid containers of the device, and measuring impedance over one ormore arrays of the device. Methods of performing cell assays usingimpedance measurement devices can be found in parent U.S. patentapplication Ser. No. 10/705,447, herein incorporated by reference forits disclosure of methods of using impedance measurement devices, aswell as in Sections D and E of the present application.

Cell-Substrate Impedance Measurement Systems

In another aspect, the present invention is directed to a cell-substrateimpedance measurement system comprising a) at least one multiple-wellcell-substrate impedance measuring device, in which at least two of themultiple wells comprise an electrode array at the bottom of the well; b)an impedance analyzer electronically connected to the multiple-wellcell-substrate impedance measuring device; c) a device station capableof engaging the one or more multiple-well devices and comprisingelectronic circuitry capable of selecting and connecting electrodearrays within any of the multiple wells to the impedance analyzer; andd) a software program connected to the device station and impedanceanalyzer to control the device station and perform data acquisition anddata analysis from the impedance analyzer.

In a cell-substrate impedance measurement system of the presentinvention, the impedance analyzer engages connection pads of one or moremulti-well devices to measure impedance. A cell-substrate measurementsystem can be used to efficiently and simultaneously perform multipleassays by using circuitry of the device station to digitally switch fromrecording from measuring impedance over an array in one well tomeasuring impedance over an array in another well.

A multiple-well cell-substrate impedance measuring device in a system ofthe present invention can be any multiple-well cell-substrate impedancemeasuring device in which at least two of the multiple wells comprise anelectrode array at the bottom of the well, and in which at least two ofthe multiple wells comprise an electrode array are individuallyaddressed. A device used in a system of the present invention, whenconnected to an impedance analyzer, can measure differences in impedancevalues that relate to cell behavior. For example, a cell-substrateimpedance measuring device used in a system of the present invention canmeasure differences in impedance values when cells are attached to theelectrode array and when cells are not attached to the electrode array,or can detect differences in impedance values when the number, type,activity, adhesiveness, or morphology of cells attached to theelectrode-comprising surface of the apparatus changes.

Preferred devices that can be part of a cell-substrate impedancemonitoring system can be those described in parent U.S. patentapplication Ser. No. 10/705,447, herein incorporated by reference fordisclosure of cell-substrate impedance monitoring devices that compriseelectrode arrays, including disclosure of their design, composition, andmanufacture. Preferred devices that can be part of a cell-substrateimpedance monitoring system can also be those described in the presentapplication.

Preferably a multi-well device of a system of the present inventioncomprises between 4 and 1,536 wells, some or all of which can compriseelectrode arrays. In some embodiments of the present invention, a devicestation can comprise one or more platforms or one or more slots forpositioning one or more multiwell devices. The one or more platforms orone or more slots can comprise sockets, pins or other devices forelectrically connecting the device to the device station. The devicestation preferably can be positioned in a tissue culture incubatorduring cell impedance measurement assays. It can be electricallyconnected to an impedance analyzer and computer that are preferablylocated outside the tissue culture incubator.

The device station comprises electronic circuitry that can connect to animpedance monitoring device and an impedance analyzer and electronicswitches that can switch on and off connections to each of the two ormore electrode arrays of the multiwell devices used in the system. Theswitches of the device station are controlled by a software program. Thesoftware program directs the device station to connect arrays of thedevice to an impedance analyzer and monitor impedance from one or moreof the electrode arrays. During impedance monitoring, the impedanceanalyzer can monitor impedance at one frequency or at more than onefrequency. Preferably, impedance monitoring is performed at more thanone time point for a given assay, and preferably, impedance is monitoredat least three time points. The device station can connect individualarrays of a device to an impedance analyzer to monitor one, some, or allof the arrays of a device for a measurement time point. The switches ofthe device station allow the selected individual arrays to be monitoredin rapid succession for each desired monitoring time point. Eachmonitoring time point is in fact a narrow time frame (for example fromless than one second to minutes) of measurement in the assay duringwhich impedance monitoring is performed. In some preferred embodimentsof the present invention, the device station software is programmable todirect impedance monitoring of any of the wells of the device thatcomprise arrays at chosen time intervals.

The software of the impedance monitoring system can also store anddisplay data. Data can be displayed on a screen, as printed data, orboth. Preferably the software can allow entry and display ofexperimental parameters, such as descriptive information including cellstypes, compound concentrations, time intervals monitored, etc.

Preferably, the software can also analyze impedance data. In preferredembodiments, the software can calculate a cell index for one or moretime points for one or more wells of the multiwell device.

FIG. 4 shows one design of a 16× device station with 6 16 well devicesconnected to the station. The device station has six individual slotsfor six devices, in which each slot comprises a zero-insertion forcesocket (416) with connection indicators (418). When a device is properlyengaged with the device station, a light (418) will be on because thelight indicator (418) will be connected through circuit lines on the PCBof the device to an electrical power source. The station compriseselectronic switches that can be switched on (connected) or off(disconnected) digitally to connect electrode arrays in individual wellsto an impedance analyzer.

FIG. 5 shows a 96 well device station with that can engage the 96-welldevice depicted in FIG. 3. The station uses POGO pins (520) to connectto the connection lines on small printed-circuit-boards (the device hassmall PCBs connected to connection pads on the edges of the device withmetal clips. The POGO-pins (520) are connected a circuitry inside thedevice station. The circuitry comprises electronic switches that can beswitched on (connected) or off (disconnected) digitally to connectelectrode arrays in those electrode-containing wells to an impedanceanalyzer.

FIG. 6 shows different pages from real-time cell electronic sensingsoftware, illustrating the entry of experimental parameters that can beentered and the display of results of data analysis. A) An experimentalnote page allows the recording of key information about the experimentby the experimenter, such as the goals and procedures of the experiment.(B) An experimental layout page allows the recording of cells, cellnumber, compound and compound concentration added into each well. (C) Atest time setting page allows for the recording and control of timeintervals used for performing cell-substrate impedance measurement andmultiple experimental steps each having different time interval valuesand different length times can be setup. (D) A cell index page is aresult page where the software automatically update the measured andderived cell index values for all wells that are under test after thecompletion of each measurement at predetermined time interval as setupby the Test time setting page. (E) An experimental data plot page allowsfor flexible plotting and organization of experimental data.

C. Methods for Calculating Cell Index

Based on the dependent relationship between the measured impedance, cellnumber (more accurately, the viable cell number, or attached cellnumber) and cell attachment status, it is possible to derive a so-called“cell number index” or “cell index” from the measured impedancefrequency spectra that provides a useful index for quantitating andcomparing cell behavior in the impedance-based assays of the presentinvention. In some applications of the present invention, “cell index”in the present application is the same as “cell number index” in PCTApplication No. PCT/US03/22557, entitled “IMPEDANCE BASED DEVICES ANDMETHODS FOR USE IN ASSAYS”, filed on Jul. 18, 2003 and in U.S. patentapplication Ser. No. 10/705,447, entitled “IMPEDANCE BASED DEVICES ANDMETHODS FOR USE IN ASSAYS,” filed on Nov. 10, 2003. U.S. patentapplication Ser. No. 10/705,447 and PCT Application No. PCT/US03/22557are hereby incorporated by reference for the discussions and disclosuresof cell index and cell number index they contain.

Various methods for calculating such a cell number index can be used,some of which are novel methods disclosed herein.

The present invention provides several methods of calculating cell indexnumbers for cells attached to two or more essentially identical arraysof a cell-substrate impedance device, where the cells are monitored forimpedance changes. In preferred embodiments of the present invention,the methods calculate cell index number with better accuracy thanprevious methods of calculating cell index for cells on two or morearrays of a cell-substrate monitoring device. In some preferred methodsof the present invention, methods of calculating a cell index rely onnovel methods for calculating the resistances of electrical tracesleading to two or more essentially identical arrays. The presentinvention therefore also includes methods of calculating resistances ofelectrical traces leading to two or more essentially identical arrays ona substrate.

By “essentially identical electrode arrays” or “essentially identicalarrays” is meant that the dimensions and arrangement of electrodes,electrode structures, and electrode elements is the same for thereferenced arrays. Thus, two essentially identical electrode arrays willhave electrode structures of the same dimensions (length, width,thickness), where the electrode structures have the same number ofelectrode elements, and the arrangement of electrode structures andelectrode elements in each array are the same. By arrangement is meantthe distance between structures or elements (gap width), their physicalposition with respect to one another, and their geometry (angles, degreeof curvature, circle-on-line or castellated geometries, etc.), includingthe same features of any electrode buses that may be connected toelectrode structures or electrode elements. Electrodes of essentiallyidentical arrays also comprise the same materials. For the purposes ofcalculating trace resistances and cell index number, a substrate canhave any number of essentially identical arrays.

The following discussion provides novel methods of calculating cellindex of cells adhered to arrays of a cell-substrate impedancemonitoring device and novel methods for the calculation of theresistances of the electrical connection traces leading to two or moreelectrode arrays of a cell-substrate impedance monitoring device.

Impedance (Z) has two components, namely the resistance Rs and reactanceXs. Mathematically, the impedance Z is expressed as follows,Z=Rs+jXs,

where j=√{square root over (−1)}, depicting that for the (serial)reactance component Xs, the voltage applied over it is 90 degreephased-out from the current going through it. For the (serial)resistance, the voltage applied over it is in phase with the currentgoing through it. As it is well-known in electronic and electricalengineering, the impedance can also be expressed in terms of parallelresistance Rp and parallel reactance Xp, as follows,Z=Rp*(jXp)/(Rp+jXp),where j=√{square root over (−1)}. Nevertheless, these expressions(serial resistance and serial reactance, or parallel resistance andparallel reactance) are equivalent. Those who are skilled in electricaland electronic engineering can readily derive one form of expressionfrom the parameter values in the other expression. For the sake ofclarity and consistency, the description and discussion in the presentinvention utilizes the expression of serial resistance and serialreactance. For simplicity, serial resistance and serial reactance aresimply called resistance and reactance.

As described in U.S. patent application Ser. No. 10/705,447, entitled“Impedance based devices and methods for use in assays”, filed on Nov.10, 2003 and PCT application number PCT/US03/22557, entitled “Impedancebased devices and methods for use in assays”, filed on Jul. 18, 2003,both of which are herein incorporated by reference for disclosuresrelating to cell-substrate impedance monitoring, monitoringcell-substrate impedance for detection or measurement of change inimpedance can be done by measuring impedance in any suitable range offrequencies. For example, the impedance can be measured in a frequencyrange from about 1 Hz to about 100 MHz. In another example, theimpedance can be measured in a frequency range from about 100 Hz toabout 2 MHz. The impedance is typically a function of the frequency,i.e., the impedance values change as frequency changes. Monitoringcell-substrate impedance can be done either in a single frequency ormultiple frequencies. If the impedance measurement is performed atmultiple frequencies, then a frequency-dependent impedance spectrum isobtained—i.e., there is an impedance value at each measured frequency.As mentioned above, the impedance has two components—a resistancecomponent and a reactance component. A change in either resistancecomponent or reactance component or both components can constitute achange in impedance.

As described in the U.S. patent application Ser. No. 10/705,447,entitled “Impedance based devices and methods for use in assays”, filedon Nov. 10, 2003 and PCT application number PCT/US03/22557, entitled“Impedance based devices and methods for use in assays”, filed on Jul.18, 2003, herein incorporated by reference for disclosure of methods ofmeasuring electrical impedance, the method for the measurement ofelectrical (or electronic) impedance is achieved by, (1) applying avoltage between or among said electrodes at a given frequency (ormultiple frequencies, or having specific voltage waveform) andmonitoring the electrical current through said electrodes at thefrequency (or multiple frequencies, or having specific waveform),dividing the voltage amplitude value by the current amplitude value toderive the impedance value; (2) applying an electric current of a singlefrequency component (or multiple frequencies or having specific currentwave form) through said electrodes and monitoring the voltage resultedbetween or among said electrodes at the frequency (or multiplefrequencies, or having specific waveform), dividing the voltageamplitude value by the current amplitude value to derive the impedancevalue; (3) other methods that can measure or determine electricimpedance. Note that in the description above of “dividing the voltageamplitude value by the current amplitude value to derive the impedancevalue”, the “division” is done for the values of current amplitude andvoltage amplitude at same frequencies. As it is well-known in electricaland electronic engineering, in such calculations (e.g. divisionsmentioned above), the current amplitude and voltage amplitude areexpressed in the form of complex numbers, which take into account of howbig the current and the voltage are and what the phase differencebetween the sinusoidal waves of the current and the voltage is.Similarly, the impedance value is also expressed in a complex form,having both resistance and reactance component, as shown in equationsabove.

As described in the U.S. patent application Ser. No. 10/705,447,entitled “Impedance based devices and methods for use in assays”, filedon Nov. 10, 2003 and PCT application number PCT/US03/22557, entitled“Impedance based devices and methods for use in assays”, filed on Jul.18, 2003, both incorporated herein by reference for disclosure relatingto Cell Index or Cell Number Index, the measured cell-substrateimpedance can be used to calculate a parameter termed Cell Index or CellNumber Index. Various methods for calculating such a cell number indexcan be used based on the changes in resistance or reactance when cellsare attached to the electrode structures with respect to the cases nocells are attached to the electrode structures. The impedance(resistance and reactance) of the electrode structures with no cellattached but with same cell culture medium over the electrode structuresis sometimes referred as baseline impedance. The baseline impedance maybe obtained by one or more of the following ways: (1) the impedancemeasured for the electrode structures with a cell-free culture mediumintroduced into the well containing the electrode structures, whereinthe culture medium is the same as that used for the impedancemeasurements for the condition where the cell attachment is monitored;(2) the impedance measured shortly (e.g. 10 minutes) after thecell-containing medium was applied to the wells comprising the electrodestructures on the well bottom (during the short period aftercell-containing medium addition, cells do not have enough time to attachto the electrode surfaces. The length of this short-period may depend oncell type and/or surface treatment or modification on the electrodesurfaces); (3) the impedance measured for the electrode structures whenall the cells in the well were killed by certain treatment (e.g.high-temperature treatment) and/or reagents (e.g. detergent) (for thismethod to be used, the treatment and/or reagents should not affect thedielectric property of the medium which is over the electrodes).

In one example (A), the cell index or cell number index can becalculated by:

-   -   (A1) at each measured frequency, calculating the resistance        ratio by dividing the resistance of the electrode arrays when        cells are present and/or attached to the electrodes by the        baseline resistance,    -   (A2) finding or determining the maximum value in the resistance        ratio over the frequency spectrum,    -   (A3) and subtracting one from the maximum value in the        resistance ratio.

Using a mathematically formula, Cell Index is derived as

$\begin{matrix}{{{Cell}\mspace{14mu}{Index}} = {\max\limits_{{i = 1},2,{\ldots\mspace{11mu} N}}\left( {\frac{R_{cell}\left( f_{i} \right)}{R_{b}\left( f_{i} \right)} - 1} \right)}} & (4)\end{matrix}$Where N is the number of the frequency points at which the impedance ismeasured. For example, if the frequencies used for the measurements areat 10 kHz, 25 kHz and 50 kHz, then N=3, f₁=10 kHz, f₂=25 kHz, f₃=50 kHz.R_(cell)(f_(i)) is the resistance (cell-substrate resistance) of theelectrode arrays or electrode structures when the cells are present onthe electrodes at the frequency f_(i) and R_(b)(f_(i)) is the baselineresistance of the electrode array or structures at the frequency f_(i).

In this case, a zero or near-zero “cell index or cell number index”indicates that no cells or very small number of cells are present on orattached to the electrode surfaces. A higher value of “cell numberindex” indicates that, for same type of the cells and cells undersimilar physiological conditions, more cells are attached to theelectrode surfaces. A higher value of “cell index” may also indicatethat, for same type of the cells and same number of the cells, cells areattached better (for example, cells spread out more, or cell adhesion tothe electrode surfaces is stronger) on the electrode surfaces.

In another example (B), the cell number index can be calculated by:

-   -   (B1) at each measured frequency, calculating the reactance ratio        by dividing the reactance of the electrode arrays when cells are        present on and/or attached to the electrodes by the baseline        reactance,    -   (B2) finding or determining the maximum value in the reactance        ratio over the frequency spectrum,    -   (B3) and subtracting one from the maximum value in the        resistance ratio.

In this case, a zero or near-zero “cell number index” indicates that nocells or very small number of cells are present on or attached to theelectrode surfaces. A higher value of “cell number index” indicatesthat, for same type of the cells and cells under similar physiologicalconditions, more cells are attached to the electrode surfaces.

In yet another example (C), the cell index can be calculated by:

-   -   (C1) at each measured frequency, subtracting the baseline        resistance from the resistance of the electrode arrays when        cells are present or attached to the electrodes to determine the        change in the resistance with the cells present relative to the        baseline resistance;    -   (C2) then finding or determining the maximum value in the change        of the resistance.

In this case, “cell-number index” is derived based on the maximum changein the resistance across the measured frequency range with the cellspresent relative to the baseline resistance. This cell index would havea dimension of ohm.

In yet another example (D), the cell index can be calculated by:

-   -   (D1) at each measured frequency, calculating the magnitude of        the impedance (equaling to √{square root over (R_(s) ²+X_(s)        ²)}, where R_(s) and X_(s) are the serial resistance and        reactance, respectively).    -   (D2) subtracting the magnitude of the baseline impedance from        the magnitude of the impedance of the electrode arrays when        cells are present or attached to the electrodes to determine the        change in the magnitude of the impedance with the cells present        relative to the baseline impedance;    -   (D3) then finding or determining the maximum value in the change        of the magnitude of the impedance.

In this case, “cell-number index” is derived based on the maximum changein the magnitude of the impedance across the measured frequency rangewith the cells present relative to the baseline impedance. This cellindex would have a dimension of ohm.

In yet another example (E), the index can be calculated by:

-   -   (E1) at each measured frequency, calculating the resistance        ratio by dividing the resistance of electrode arrays when cells        are present or attached to the electrodes by the baseline        resistance,    -   (E2) then calculating the relative change in resistance in each        measured frequency by subtracting one from the resistance ratio,    -   (E3) then integrating all the relative-change value (i.e.,        summing together all the relative-change values at different        frequencies).

In this case, “cell-number index” is derived based on multiple-frequencypoints, instead of single peak-frequency like above examples. Again, azero or near-zero “cell number index” indicates that on cells arepresent on the electrodes. A higher value of “cell number index”indicates that, for same type of the cells and cells under similarphysiological conditions, more cells are attached to the electrodes.

In yet another example (F), the cell index can be calculated by:

-   -   (F1) at each measured frequency, subtracting the baseline        resistance from the resistance of the electrode arrays when        cells are attached to the electrodes to determine the change in        the resistance with the cells present relative to the baseline        impedance; (here the change in the resistance is given by        ΔR(f_(i))=R_(s-cell)(f_(i))−R_(s-baseline)(f_(i)) for the        frequency f_(i), R_(s-cell) and R_(s-baseline) are the serial        resistances with the cells present on the electrode array and        the baseline serial resistances, respectively);    -   (F3) analyzing the frequency dependency of the change of the        resistance to derive certain parameters that can quantify such        dependency. In one example, such parameters can be calculated as

$\sqrt{\sum\limits_{i}\left\lbrack {\Delta\;{R\left( f_{i} \right)}} \right\rbrack^{2}}.$In another example, such parameter can be calculated as

$\sum\limits_{i}{{{\Delta\;{R\left( f_{i} \right)}}}.}$The parameter(s) are used as cell index or cell number index.

In this case, “cell-number index” is derived based on the analysis ofthe frequency spectrum of the change in the resistance. Depending howthe parameters are calculated, the cell index may have a dimension ofohm.

In yet another example (G), the cell index can be calculated by:

-   -   (G1) at each measured frequency, calculating the magnitude of        the impedance (equaling to √{square root over (R_(s) ²+X_(s)        ²)}, where R_(s) and X_(s) are the serial resistance and        reactance, respectively).    -   (G2) subtracting the magnitude of the baseline impedance from        the magnitude of the impedance of the electrode arrays when        cells are attached to the electrodes to determine the change in        the magnitude of the impedance with the cells present relative        to the baseline impedance; (here, the change in the magnitude of        the impedance is given by        ΔZ(f_(i))=|Z_(cell)(f_(i))|−|Z_(baseline)(f_(i))| for the        frequency f_(i), |Z_(cell)(f_(i))|=√{square root over        (R_(s-cell)(f_(i))²+X_(s-cell)(f_(i))²)}{square root over        (R_(s-cell)(f_(i))²+X_(s-cell)(f_(i))²)}, R_(s-cell) and        X_(s-cell) being the serial resistance and reactance with the        cells present on the electrode arrays, respectively,        |Z_(cell)(f_(i))| is the magnitude of the impedance of the        electrode array with cells present on the electrode arrays,        |Z_(baseline)(f_(i))| is the magnitude of the baseline impedance        of the electrode array);    -   (G3) analyzing the frequency dependency of the change of the        magnitude of the impedance to derive certain parameters that can        quantify such dependency. In one example, such parameters can be        calculated as

$\sqrt{\sum\limits_{i}\left\lbrack {\Delta\;{Z\left( f_{i} \right)}} \right\rbrack^{2}}.$In another example, such parameter can be calculated as

$\sum\limits_{i}{{{\Delta\;{Z\left( f_{i} \right)}}}.}$The parameter(s) are used as cell index or cell number index.

In this case, “cell-number index” is derived based on the analysis ofthe frequency spectrum of the change in the magnitude of the impedance.Depending how the parameters are calculated, the cell index may have adimension of ohm.

As described in the U.S. patent application Ser. No. 10/705,447,entitled “Impedance based devices and methods for use in assays”, filedon Nov. 10, 2003 and PCT application number PCT/US03/22557, entitled“Impedance based devices and methods for use in assays”, filed on Jul.18, 2003, both herein incorporated by reference for disclosure of CellIndex or Cell Number Index and its calculation, there are differentmethods for calculating the parameter termed Cell Index or Cell NumberIndex from the measured cell-substrate impedance (resistance orreactance). Cell Index or Cell Number Index is a quantitative measure ofcells in the wells under cell-substrate impedance measurement.

It is worthwhile to point out that it is not necessary to derive such a“cell number index” for utilizing the impedance information formonitoring cell conditions over the electrodes. Actually, one may chooseto directly use impedance values (e.g., at a single fixed frequency; orat a maximum relative-change frequency, or at multiple frequencies) asan indicator of cell conditions.

Still, deriving “cell index” or “cell number index” and using such indexto monitor cell conditions may have advantages. There are severaladvantages of using “cell number index” to monitor cell growth and/orattachment and/or viability conditions.

First, one can compare the performance of different electrode geometriesby utilizing such cell number index.

Secondly, for a given electrode geometry, it is possible to construct“calibration curve” for depicting the relationship between the cellnumber and the cell number index by performing impedance measurementsfor different number of cells added to the electrodes (in such anexperiment, it is important to make sure that the seeded cells havewell-attached to the electrode surfaces). With such a calibration curve,when a new impedance measurement is performed, it is then possible toestimate cell number from the newly-measured cell number index.

Thirdly, cell number index can also be used to compare different surfaceconditions. For the same electrode geometry and same number of cells, asurface treatment given a larger cell number index indicates a betterattachment for the cells to the electrode surface and/or better surfacefor cell attachment.

As shown above, for some methods of calculating cell index or cellnumber index, it is important to know the impedance (resistance and/orreactance) of the electrode structures with and without cells present onthem. Based on the equation (1), the impedance of the electrode array(with or without cells present on the electrodes) is given byZ _(electrode-array) =Z _(total) −Z _(trace) −Z _(switch)  (5)

Where Z_(switch) is the impedance of electronic switch at its on stage,Z_(trace) is the impedance of the electrical connection traces (orelectrical conductive traces) on the substrate between the connectionpads and the electrode buses, Z_(total) is the total impedance measuredat the impedance analyzer. By choosing electronic switches with goodquality, it is possible to have all the electronic switches have aconsistent on-impedance (mainly resistance). For example, theon-resistance of electronic switches can be about 3 ohm (+/−10%) withthe on reactance being negligible (for example, less than 0.2 ohm in thefrequency range of interest). Thus, if the trace impedance is determinedor calculated, then formula (5) can be used to calculate the impedanceof the electrode arrays with or without cells present.

A method is invented in the present application to determine theimpedance of electrical conductive (electrical connection) traces(mainly trace resistance, trace reactance is very small for the thinconductive film trace) based on the relationships among two or moreessentially identical arrays on a cell-substrate impedance monitoringdevice. In the following, the four electrode arrays A, B, C and D asindicated in FIG. 1A, are used to illustrate this method. The electricalreactance (serial reactance) of the electronic switches and theelectrical reactance (serial reactance) of the electrical connectiontraces are small as compared with the corresponding electricalresistances (serial resistances). Thus, we focus on the analysis of theresistance of the electrical connection traces. The impedance determinedfrom the impedance analyzer does contain both resistance (serialresistance, R_(total)) and reactance (serial reactance). For theelectrode arrays A-D, the measured total resistance R_(total), theresistance (R_(trace)) of electrical conductive (connection) trace, theswitch resistance (R_(switch)) and the resistance (R_(e-array)) of theelectrode array satisfy the following equations:R _(e-array-A) =R _(total-A) −R _(trace-A) −R _(switch-A)  (6A)R _(e-array-B) =R _(total-B) −R _(trace-B) −R _(switch-B)  (6B)R _(e-array-C) =R _(total-C) −R _(trace-C) −R _(switch-C)  (6C)R _(e-array-D) =R _(total-D) −R _(trace-D) −R _(switch-D)  (6D)With chosen electronic switches having consistent switch-on resistance,R_(switch-A), R_(switch-B), R_(switch-C) and R_(switch-D) have verysimilar values and can be assumed to be the same, R_(switch). Thus, inabove equations, the known parameters are R_(total-A), R_(total-B),R_(total-C), and R_(total-D), and R_(switch-A), R_(switch-B),R_(switch-C) and R_(switch-D), and there are eight unknown parametersR_(e-array-A), R_(e-array-B), R_(e-array-C), and R_(e-array-D), andR_(trace-A), R_(trace-B), R_(trace-C) and R_(trace-D). It is impossibleto solve these equations for the eight unknown variables from these fourequations directly. Additional relationships between these variables areneeded to solve for them. Each trace resistance (R_(trace-A),R_(trace-B), R_(trace-C) and R_(trace-D)) depends on the metal film typeused, and the geometry of the trace such as the how many rectangularsegments the trace has, the film thickness(es) of the segments, thewidth(s) of the segments, the length(s) of the segment(s). For example,

$R_{{trace} - A} = {\sum\limits_{i = 1}^{N}{\rho\frac{L_{A - i}}{t_{A - i}*d_{A - i}}}}$where N is the number of the segments of the trace-A, t_(A-i), d_(A-i)and L_(A-i) is the thickness, width and length of the i-th segment ofthe traces for the electrode array A, and ρ is the resistivity of thethin film. The equation here applies to the film comprising a singletype of metal. The equation can be readily modified to be applicable tothe film comprising two or more metal types (e.g. gold film overchromium adhesion layer).

If the film thickness is reasonably uniform (for example, less than 10%in thickness variation) across the substrate, then the relationshipamong the trace resistances is simply determined by the pre-determinedgeometrical shapes (e.g. the length, width of the segments). Forexample, it would be straightforward to calculate the ratio α_(A-D)between the resistance of the electrically conductive traces for theelectrode array A to the resistance of the electrically conductivetraces for the electrode array D as below, where the film thickness isassumed to be the same everywhere on these traces and the resistivity isalso the same everywhere on these traces,

$\begin{matrix}{\alpha_{A - D} = {\frac{R_{{trace}\_ A}}{R_{{trace}\_ D}} = {\frac{\sum\limits_{i = 1}^{N}{\rho\frac{L_{A - i}}{t_{A - i}*d_{A - i}}}}{\sum\limits_{i = 1}^{M}{\rho\frac{L_{D - i}}{t_{D - i}*d_{D - i}}}} = {\frac{\sum\limits_{i = 1}^{N}\frac{L_{A - i}}{d_{A - i}}}{\sum\limits_{i = 1}^{M}\frac{L_{D - i}}{d_{D - i}}}.}}}} & (8)\end{matrix}$Similarly, one can determine the ratio α_(B-D) and α_(C-D) based on thepre-determined geometrical relationships for the traces of the electrodearrays B, C and D. Note that above equations can be similarly derivedfor the cases where the thin film in these traces comprises more thanone metal type. Thus, based on the equalitiesR _(switch-A) =R _(switch-B) =R _(switch-C) =R _(switch-D) =R_(switch)  (9A)R _(trace-A)=α_(A-D) ·R _(trace-D),  (9B)R _(trace-B)=α_(B-D) ·R _(trace-D),  (9C)and R _(trace-C)=α_(C-D) ·R _(trace-D)  (9D)equations (6A)-(6D) can be re-written in the following format:R _(e-array-A) =R _(total-A)−α_(A-D) ·R _(trace-D) −R _(switch)  (10A)R _(e-array-B) =R _(total-B)−α_(B-D) ·R _(trace-D) −R _(switch)  (10B)R _(e-array-C) =R _(total-C)−α_(C-D) ·R _(trace-D) −R _(switch)  (10C)R _(e-array-D) =R _(total-D) −R _(trace-D) −R _(switch-D)  (10D)For equations (10A) through (10D), there are five unknown variables,R_(e-array-A), R_(e-array-B), R_(e-array-C), and R_(e-array-D) andR_(trace-D). Mathematically, these unknown variables cannot bedetermined from these equations. Additional information is needed tosolve for these variables R_(e-array-A), R_(e-array-B), R_(e-array-C),and R_(e-array-D) and R_(trace-D).

One approach is invented and described in the present invention. In thisapproach, same biological or chemical solutions or suspensions areapplied to the electrode-arrays A through D. Because the electrodearrays A through D have essentially identical electrode structures, theelectrode array resistances R_(e-array-A), R_(e-array-B), R_(e-array-C)and R_(e-array-D) should be of same, or very similar value for such acondition when all the electrode arrays are exposed to the samebiological or chemical solutions or suspensions, i.e.:R_(e-array-A)≈R_(e-array-B)≈R_(e-array-C)≈R_(e-array-D). If we assumethe averaged electrode array resistance is R_(e-array), then theseapproximate relationship existsR_(e-array-A)≈R_(e-array-B)≈R_(e-array-C)≈R_(e-array-D)≈R_(e-array).Thus, equations (10A-10D) can be changed to the following:R _(e-array) ≈R _(total-A)−α_(A-D) ·R _(trace-D) −R _(switch)  (11A)R _(e-array) ≈R _(total-B)−α_(B-D) ·R _(trace-D) −R _(switch)  (11B)R _(e-array) ≈R _(total-C)−α_(C-D) ·R _(trace-D) −R _(switch)  (11C)R _(e-array) ≈R _(total-D) −R _(trace-D) −R _(switch-D)  (11D)

Thus, we would need to find R_(trace-D) and R_(e-array) that satisfy theabove approximate equality as close as possible. One mathematicalapproach is to find R_(trace-D) and R_(e-array) that would result in theminimum value for the following expression—an expression that quantifiesthe differences between the two sides of the approximate equality in(11A, 11B, 11C and 11D),F(R _(trace-D) ,R _(e-array))=[R _(e-array)−(R _(total-A)−α_(A-D) R_(trace-D) −R _(switch))]² +[R _(e-array)−(R _(total-B)−α_(B-D) R_(trace-D) −R _(switch))]² +[R _(e-array)−(R _(total-C)−α_(C-D) R_(trace-D) −R _(switch))]² +[R _(e-array)−(R _(total-D) −R _(trace-D) −R_(switch))]²  (12)The expression F(R_(trace-D),R_(e-array)) is the sum of thesquared-differences between the two-sides of the approximate equality in(11A, 11B, 11C and 11D). The smaller F(R_(trace-D),R_(e-array)), thecloser the two sides of the approximate equality (11A, 11B, 11C and11D). Thus, values of R_(trace-D) and R_(e-array) that result in theminimum value of F(R_(trace-D),R_(a-array)) should be determined.Mathematical approach involves in the calculation of the first orderderivative of F(R_(trace-D),R_(e-array)) to R_(trace-D) and toR_(e-array) and let such first order derivatives equal to zero. Thevalues of R_(trace-D) and R_(e-array) that result in zero for thesefirst-order-derivatives are those that result in the minimum value ofF(R_(trace-D),R_(e-array)). The first order derivatives are as follows:

$\begin{matrix}{{\frac{\partial\left\lfloor {F\left( {R_{{trace} - D},R_{e - {aaray}}} \right)} \right\rfloor}{\partial R_{{trace} - D}} = {{{2 \cdot \alpha_{A - D} \cdot \left\lbrack {R_{e - {array}} - \left( {R_{{total} - A} - {\alpha_{A - D}R_{{trace} - D}} - R_{switch}} \right)} \right\rbrack} + {2 \cdot \alpha_{B - D} \cdot \left\lfloor {R_{e - {array}} - \left( {R_{{total} - B} - {\alpha_{B - D}R_{{trace} - D}} - R_{switch}} \right)} \right\rfloor} + {2 \cdot \alpha_{C - D} \cdot \left\lfloor {R_{e - {array}} - \left( {R_{{total} - C} - {\alpha_{C - D}R_{{trace} - D}} - R_{switch}} \right)} \right\rfloor} + {2 \cdot \left\lfloor {R_{e - {array}} - \left( {R_{{total} - D} - R_{{trace} - D} - R_{switch}} \right)} \right\rfloor}} = 0}};} & \left( {13A} \right) \\{\frac{\partial\left\lfloor {F\left( {R_{{trace} - D},R_{e - {aaray}}} \right)} \right\rfloor}{\partial R_{e - {array}}} = {{{2 \cdot \left\lbrack {R_{e - {array}} - \left( {R_{{total} - A} - {\alpha_{A - D}R_{{trace} - D}} - R_{switch}} \right)} \right\rbrack} + {2 \cdot \left\lfloor {R_{e - {array}} - \left( {R_{{total} - B} - {\alpha_{B - D}R_{{trace} - D}} - R_{switch}} \right)} \right\rfloor} + {2 \cdot \left\lfloor {R_{e - {array}} - \left( {R_{{total} - C} - {\alpha_{C - D}R_{{trace} - D}} - R_{switch}} \right)} \right\rfloor} + {2 \cdot \left\lfloor {R_{e - {array}} - \left( {R_{{total} - D} - R_{{trace} - D} - R_{switch}} \right)} \right\rfloor}} = 0.}} & \left( {13B} \right)\end{matrix}$Equations (13A) and (13B) can be re-written asR _(e-array)·[α_(A-D)+α_(B-D)+α_(C-D)+1]+R _(trace-D)·└α_(A-D) ²+α_(B-D)²+α_(C-D) ²+1┘=α_(A-D) ·[R _(total-A) −R _(switch)]+α_(B-D) ·[R_(total-B) −R _(switch)]+α_(C-D) ·[R _(total-C) −R _(switch) ]+[R_(total-D) −R _(switch)]  (14A)4·R _(e-array) +R _(trace-D)·[α_(A-D)+α_(B-D)+α_(C-D)+1]=[R _(total-A)−R _(switch) ]+[R _(total-B) −R _(switch) ]+[R _(total-C) −R _(switch)]+[R _(total-D) −R _(switch)]  (14B)Thus, we can solve for R_(trace-D) as follows:

$\begin{matrix}{R_{{trace} - D} = \frac{{4 \cdot S_{1}} - {A_{11} \cdot S_{2}}}{{4 \cdot A_{12}} - {A_{11} \cdot B_{12}}}} & (15)\end{matrix}$where

-   -   A₁₁=[α_(A-D)+α_(B-D)+α_(C-D)+1];    -   A₁₂=└α_(A-D) ²+α_(B-D) ²+α_(C-D) ²+1┘;    -   S₁=α_(A-D)·[R_(total-A)−R_(switch)]+α_(B-D)·[R_(total-B)−R_(switch)]+α_(C-D)·[R_(total-C)−R_(switch)]+[R_(total-D)−R_(switch)];    -   B₁₂=[α_(A-D)+α_(B-D)+α_(C-D)+1];    -   S₂=[R_(total-A)−R_(switch)]+[R_(total-B)−R_(switch)]+[R_(total-C)−R_(switch)]+[R_(total-D)−R_(switch)].        Thus, with the determined R_(trace-D), the trace resistances of        R_(trace-A), R_(trace-B), and R_(trace-C) can be calculated        using equations (9B), (9C) and (9D). Furthermore, the electrode        array resistance R_(e-array-A), R_(e-array-B), R_(e-array-C) and        R_(e-array-D) can be calculated from the measured resistance        R_(total-A), R_(total-B), R_(total-C) and R_(total-D)        respectively using equations (10A), (10B), (10C) and (10D).

Thus, one aspect of the present invention is directed to a method ofcalculation of the resistances of the electrical connection traces sfrom the measured, total resistances for two or more essentiallyidentical electrode arrays (such as, for example arrays A-D in FIG. 1A),comprising the following steps:

-   -   (1) exposing the electrode arrays to the solutions having same        or similar solutions or suspensions;    -   (2) with an impedance analyzer or impedance measurement circuit,        measuring the resistance (serial resistance) for each electrode        array, such resistance being the sum of the resistance of        electronic switches, the resistance of the electrical connection        traces between the connection pads and the electrode structures        (for example, between the connection pads and the electrode        buses, for the electrode structures in FIG. 1A), and the        resistance of the electrode array with the solutions or        suspensions present;    -   (3) solving for the resistances of electrical connection traces        using equation (15) and equations (9B), (9C) and (9D), noting in        the calculation with equation (15), the geometrical        relationships between the electrode arrays are used to determine        the factor α_(A-D), α_(B-D) and α_(C-D).

Another aspect of the present invention is directed to a method ofcalculating the resistance of the electrode arrays from the measured,total electrode resistances for two or more essentially identicalelectrode arrays (such as, for example arrays A-D in FIG. 1A) if thesame or similar solutions or suspensions are added to be in contact withthe electrode assays, comprising the following steps:

-   -   (1) exposing the electrode arrays to the solutions having same        or similar solutions or suspensions;    -   (2) with an impedance analyzer or impedance measurement circuit,        measuring the resistance (serial resistance) for each electrode        array, such resistance being the sum of the resistance of        electronic switches, the resistance of the electrical connection        traces between the connection pads and the electrode structures        (for example, between the connection pads and the electrode        buses, for the electrode structures in FIG. 1A) and the        resistance of the electrode arrays with the solutions or        suspensions present;    -   (3) solving for the resistances of electrical connection traces        using equation (15) and equations (9B), (9C) and (9D), noting in        the calculation with equation (15), the geometrical        relationships between the electrode arrays are used to determine        the factor α_(A-D), α_(B-D) and α_(C-D);    -   (4) calculating the resistances of the electrode arrays using        equations (10A, 10B, 10C and 10D)).

In many applications, the solutions or suspensions (for example, cellsuspension) applied to each electrode array may have differentcompositions. For example, cell suspensions of different cell numbersmay be used so that the suspensions applied to each electrode array arequite different. Under such cases, the determination of the resistanceof the electrode arrays with the cells present would require thedetermination of the resistance of the electrical connection traces byperforming a “reference run” or “calibration run” in which the electrodearrays are exposed to a same, reference solution. From the “referencerun”, the resistances of the electrical connection traces can bedetermined. In a separate test, the electrode arrays are exposed to thesolutions or cell suspensions of interest and the resistances for theelectrode arrays under such conditions are measured with an impedanceanalyzer or impedance measuring circuit. The resistance of the electrodearrays with such cell suspensions present can be determined (orcontinuously determined) from the measured resistance by subtracting thesum of the resistance of the electronic switches and the resistance ofthe electrical connection traces for corresponding electrode arrays fromthe measured resistances.

Thus, another aspect of the present invention is directed to a method ofcalculating the resistance of the electrode arrays from the totalelectrical resistances measured at an impedance analyzer for essentiallyidentical electrode arrays (such as electrode arrays A-D in FIG. 1A usedas an example) if different solutions or suspensions of interest areapplied to the electrode assays, comprising the following steps:

-   -   (1) exposing the electrode arrays to the solutions having same        or similar solutions or suspensions (reference solutions);    -   (2) with an impedance analyzer or impedance measurement circuit,        measuring the resistance (serial resistance) for each electrode        array, such resistance being the sum of the resistance of        electronic switches, the resistance of the electrical connection        traces between the connection pads and the electrode structures        (for example, between the connection pads and the electrode        buses, for the electrode structures in FIG. 1A) and the        resistance of the electrode arrays with the reference solutions        present;    -   (3) solving for the resistances of electrical connection traces        using equation (15) and equations (9B), (9C) and (9D), noting in        the calculation with equation (15), the geometrical        relationships between the electrode arrays of FIG. 1A are used        to determine the factor α_(A-D), α_(B-D) and α_(C-D);    -   (4) applying the solutions or suspensions of interest to each        electrode array; and with an impedance analyzer or impedance        measurement circuit, measuring the resistance (serial        resistance) of each electrode array, such resistance being the        sum of the resistance of electronic switches, the resistance of        the electrical connection traces between the connection pads and        the electrode structures, the resistance of the electrode arrays        with the solutions or suspensions of the interest present,    -   (5) Calculating the resistance of the electrode arrays using        equations (10A), (10B), (10C) and (10D) by subtracting the        electronic switch resistances and the resistances of electrical        connection traces from the measured resistances in the step (4).

Note that in above method, the steps of exposing the electrode arrays toreference solutions for the determination of the resistances ofelectrically conductive traces (step (1), (2) and (3)) may be performedbefore or after the steps of applying the solutions or suspensions ofinterest to the electrode arrays and measuring the total electricalresistance (step (4)). For example, step (4) may be performed first.After that, the solutions or suspensions of the interest may be removedfrom the electrode array. The reference solutions can then be added tothe electrode arrays (step (1)). Step (2) and step (3) can be thenperformed to determine the resistances of electrical connection traces.Finally, Step (5) can be done.

In another approach, step (1) and (2) can be performed ahead of step(4).

Another aspect of the present invention is directed to a method ofdetermining the resistance of the electrode arrays with the cellspresent for a cell-based assay based on the total electrical resistancemeasured at an impedance analyzer for essentially identical electrodearrays. In this method, the electrode arrays are exposed to a same,reference solution (for example, a same cell culture medium that doesnot contain any cells) and electrical measurement is conducted todetermine the resistance of electrical connection traces. With theresistances of the electrical connection traces determined, electricalresistances of the electrode arrays with cell suspensions added toelectrode arrays can be calculated from the total electrical resistancesmeasured at an impedance analyzer. Such total electrical resistancewould include the resistance of the electrode arrays with cells present,the resistance of electronic switches and the resistance of electricalconnection traces. The method comprises following steps

-   -   (1) exposing the electrode arrays to the solutions having same        or similar solutions or suspensions (reference solutions);    -   (2) with an impedance analyzer or impedance measurement circuit,        measuring the resistance (serial resistance) for each electrode        array, such resistance being the sum of the resistance of        electronic switches, the resistance of the electrical connection        traces between the connection pads and the electrode structures        (for example, between the connection pads and the electrode        buses, for the electrode structures in FIG. 1A) and the        resistance of the electrode arrays with the reference solutions        present;    -   (3) solving for the resistances of electrical connection traces        using equation (15) and equations (9B), (9C) and (9D), noting in        the calculation with equation (15), the geometrical        relationships between the electrode arrays in FIG. 1A are used        to determine the factor α_(A-D), α_(B-D) and α_(C-D);    -   (4) applying the cell suspensions of interest to each electrode        array; and with an impedance analyzer or impedance measurement        circuit, measuring the resistance (serial resistance) of each        electrode array, such resistance being the sum of the resistance        of electronic switches, the resistance of the electrical        connection traces between the connection pads and the electrode        structures, the resistance of the electrode arrays with the cell        suspensions of the interest present,    -   (5) Calculating the resistance of the electrode arrays using        equations (10A), (10B), (10C) and (10D) by subtracting the        electronic switch resistances and the resistances of electrical        connection traces from the measured resistances in step (4).

Note that in above method, the steps of exposing the electrode arrays toreference solution for the determination of the electrical resistance ofelectrically conductive traces (step (1), (2) and (3)) may be performedbefore or after the steps of applying the solutions of interest or cellsuspensions of interest to the electrode arrays and measuring the totalelectrical resistance (step (4)). For example, step (4) may be performedfirst, followed by steps (1) and (2). In one approach, after step (4),the cell suspensions of the interest may be removed from the electrodearray. Then reference solutions can be added to the electrode arrays. Inanother approach, after step (4), the cells are all lysed with some celllysis solutions so that the electrodes are exposed to the same,reference solutions for the measurement and calculation of step (2) and(3). And then, step (5) is performed to determine the electricalresistance of electrode arrays with the cell suspensions of interestpresent.

The determination of the resistances of the electrical conductive tracesfor the electrode arrays that essentially identical electrode arrays maybe, or may not be, part of the monitoring of cell-substrate impedancefor cell-based assays. It depends on how the impedance data (measured ata single frequency or multiple frequencies, measured at multiple timepoints) of the electrode arrays is analyzed.

In some assays, one is interested in the relative change in theresistance or impedance of the electrode arrays with the cells presentrelative to the baseline resistance or impedance. For such cases, it ispreferred to determine the resistance (or impedance) of the electrodearrays from the total, measures resistance (or impedance) by subtractingthe resistance of the electrical conductive traces and the resistance ofelectronic switches. Thus, determination of the resistances or impedanceof the electrically conductive traces may be required.

In some other assays, one is interested in the absolute changes in theresistance (or impedance) of the electrode arrays with cells presentrelative to the baseline resistance (or impedance). In these cases, onecan directly subtract the measured resistance or impedance for thebaseline condition from the measured resistance or impedance for thecondition that the cells are present on the electrode arrays. Thecontribution of the resistance (or impedance) of the electronic switchesand the resistance (or impedance) of the electrically conductive tracesto the total measured resistance (or impedance) values is cancelled outin such subtractions. Thus, there is no need for determining theresistances of the electrically conductive traces.

In some assays, one is interested in calculating the Cell Index or CellNumber Index based on the monitored impedance values. Depending on whichmethod is used for calculating the Cell Index, it may, or may not, benecessary to determine the resistances of the electrically conductivetraces. For example, for the Cell Index calculation method (A) describedabove, the resistances of the electrically conductive traces are needed,in order to remove the effect of the resistance of the electricallyconductive traces on the analysis of the relative change of theresistance or impedance. In another example, for the Cell Indexcalculation method (F) described above, there is no need to determinethe resistances of the electrically conductive traces since the effectof the resistance of the electrically conductive traces is canceled outin the calculations.

The monitoring of the cell-substrate impedance may be or may not bebased on the change with respect to the baseline impedance (orresistance). For example, a cell-based assay is performed to assess theeffect of a test compound on the cells. One method in performing such anassay is by monitoring of the cell-substrate impedance and determiningthe change in the cell-substrate impedance before and after the additionof the test compound to the cells. The monitoring of cell-substrateimpedance can be performed at a single frequency point or multiplefrequency points, at a single time point or multiple time points afterdrug addition. For example, the impedance is first measured at a singlefrequency or multiple frequencies for the electrode arrays with thecells present just before addition of test compound. The test compoundis then added to the cells. The impedance is then measured again at thesame single frequency or multiple frequencies for the electrode arrayswith the cells after the addition of test compound. Such post-compoundaddition measurement may be performed for many time points continuouslyin a regular or irregular time intervals. The change in thecell-substrate impedances can be determined or quantified by subtractingthe impedance(s) (resistance and/or reactance) measured before additionof the test compound from the impedance(s) (resistance and/or reactance)measured after addition of the test compound. If the measurement is doneat multiple frequencies, a single parameter or multiple parameters maybe further derived for each time point after compound addition based onthe calculated change in the cell-substrate impedances. Such parametersare used to quantify the cell changes after compound addition. Suchapproaches can be used further to analyze the responses of the cells toa test compound at multiple concentrations to derive dose-dependentresponse curves.

D. Methods for Performing Real-Time Cell-Based Assays

The present invention provide cell-based assays that can be performed inreal time to assess cell proliferation, cell growth, cell death, cellmorphology, cell membrane properties (for example, size, morphology, orcomposition of the cell membrane) cell adhesion, and cell motility. Thusthe assays can be cytotoxicity assays, proliferation assays, apoptosisassays, cell adhesion assays, cell activation assays, anti-cancercompound efficacy assays, receptor-ligand binding and signaltransduction analysis, assays of cytoskeletal changes, assays of cellstructural changes (including but not limited to, changes in cellmembrane size, morphology, or composition), assays of celldifferentiation or de-differentiation, assays of cell adhesivity, assaysof cell-cell interactions, analysis of microbial and environmentaltoxins, etc. The assays are real-time assays in the sense that cellbehavior or cell condition being assayed can be assessed continuously atregular or irregular intervals. Depending on the applications, cellbehaviors, cell responses, or cell conditions being assayed can bewithin seconds to minutes of their occurrence. The cell response duringan assay can be monitored essentially continuously over a selected timeperiod. For example, a culture can be monitored every five to fifteenminutes for several hours to several days after addition of a reagent.The interval between impedance monitoring, whether impedance monitoringis performed at regular or irregular intervals, and the duration of theimpedance monitoring assay can be determined by the experimenter.

Thus, the cell-based impedance assays of the present invention avoidinadvertently biased or misleading evaluation of cell responses due tothe time point or time points chosen for sampling or assaying the cells.In addition, the assays do not require sampling of cell cultures oraddition of reagents and thus eliminate the inconvenience, delay inobtaining results, and error introduced by many assays.

Descriptions of cell-substrate monitoring and associated devices,systems and methods of use have been provided in U.S. provisionalapplication No. 60/379,749, filed on Jul. 20, 2002; U.S. provisionalapplication No. 60/435,400, filed on Dec. 20, 2002; U.S. Provisionalapplication 60/469,572, filed on May 9, 2003, PCT application numberPCT/US03/22557, entitled “Impedance based devices and methods for use inassays”, filed on Jul. 18, 2003; PCT application number PCT/US03/22537,entitled “Impedance based apparatuses and methods for analyzing cellsand particles”, filed on Jul. 18, 2003; U.S. patent application Ser. No.10/705,447, entitled “Impedance based devices and methods for use inassays”, filed on Nov. 10, 2003; U.S. patent application Ser. No.10/705,615, entitled “Impedance based apparatuses and methods foranalyzing cells and particles”, filed on Nov. 10, 2003, all incorporatedherein by reference for their disclosure of cell-substrate impedancedevices, systems, and methods of use. Additional details ofcell-substrate impedance monitoring technology is further disclosed inthe present invention.

In brief, for measurement of cell-substrate or cell-electrode impedanceusing the technology of the present invention, cell-substrate impedancemonitoring devices are used that have microelectrode arrays withappropriate geometries fabricated onto the bottom surfaces of wells suchas microtiter plate wells, or have a similar design of having multiplefluid receptacles (wells) having electrodes fabricated on their bottomsurfaces facing into the wells. Cells are introduced into the wells ofthe devices, and make contact with and attach to the electrode surfaces.The presence, absence or change of properties of cells affects theelectronic and ionic passage on the electrode sensor surfaces.

Measuring the impedance between or among electrodes provides importantinformation about biological status of cells present on the sensors.When there are changes to the biological status of the cells analogueelectronic readout signals can be measured automatically and in realtime, and can be converted to digital signals for processing and foranalysis. In a system of the present invention, a cell index can beautomatically derived and provided based on measured electrode impedancevalues. The cell index obtained for a given well reflects: 1) how manycells are attached to the electrode surfaces in this well, 2) how well(tightly or extensively) cells are attached to the electrode surfaces inthis well. Thus, the more the cells of same type in similarphysiological conditions attach the electrode surfaces, the larger thecell index. And, the better the cells attach to the electrode surfaces(e.g., the cells spread-out more to have larger contact areas, or thecells attach tighter to electrode surfaces), the larger the cell index.

In one aspect of the present invention, a method is provided forperforming cell-based assays, comprising: a) providing a cell-substrateimpedance monitoring system of the present invention; b) introducingcells into one or more wells of a device of the system, wherein at leastone of the one or more wells comprises an electrode array; and d)monitoring cell-substrate impedance of at least one of the wells thatcomprise an electrode array and cells.

The method can be used to assay cell status, where cell status includes,not limited to, cell attachment or adhesion status (e.g. the degree ofcell spread, the attachment area of a cell, the degree of tightness ofcell attachment, cell morphology) on the substrate including on theelectrodes, cell growth or proliferation status; number of viable cellsand/or dead cells in the well; cytoskeleton change and re-organizationand number of cells going through apoptosis and/or necrosis. Thecell-based assays that be performed with above methods include, but arenot limited to, cell adhesion, cell apoptosis, cell differentiation,cell proliferation, cell survival, cytotoxicity, cell morphologydetection, cell quantification, cell quality control, time-dependentcytotoxicity profiling, IgE-mediated cell activation or stimulation,receptor-ligand binding, viral and bacterial toxin mediated cellpathologic changes and cell death, detection and quantification ofneutralizing antibodies, specific T-cell mediated cytotoxic effect,cell-based assay for screening and measuring ligand-receptor binding.

In preferred embodiments of this aspect of the present invention, cellsare added to at least two wells of a device, each of which comprises anelectrode array, and impedance is monitored from at least two wells thatcomprise cells and an electrode array.

The cells used in the assay can be primary cells isolated from anyspecies or cells of cell lines. The cells can be engineered cells. Insome embodiments, different cell types are added to different wells andthe behavior of the cells is compared.

Impedance can be monitored at regular or irregular time intervals.Preferably, impedance is monitored at three or more time points,although this is not a requirement of the present invention. In oneembodiment of the above cell-based assay, the cell-substrate impedanceis monitored at regular time intervals. Impedance can be monitored atone frequency or at more than one frequency. For example, in somepreferred embodiments, impedance is monitored over a range offrequencies for each time point at which impedance is monitored.Preferably, impedance is monitored at least one frequency between about1 Hz and about 100 MHz, more preferably at least one frequency betweenabout 100 Hz and about 2 MHz.

FIG. 7 depicts results of the use of methods of the present invention tomonitor cell proliferation. In this experiment, H460 cells wereintroduced into wells of a 16 well device of a cell-substrate impedancemonitoring system of the present invention, with different wellsreceiving different initial cell seeding numbers. The device was engagedwith a device station of the system that was in a tissue cultureincubator that kept a temperature of 37 degrees C. and an atmosphere of5% CO₂. Cell-substrate impedance was monitored at 15 minute intervalsfor 125 hours. The cell index was calculated by the system for each timepoint and displayed as a function of time to give cell growth(proliferation) curves for each cell seeding number. The cell growthcurves were plotted on a log scale showing exponential growth phases andstationary phases.

FIG. 8 depicts results of real-time monitoring of cell attachment andspreading of NIH3T3 cells. The cells were seeded onto cell-substrateimpedance monitoring devices of the present invention that were coatedwith either poly-L-lysine or fibronectin. The device was engaged with adevice station that was in a tissue culture incubator that kept atemperature of 37 degrees C. and an atmosphere of 5% CO₂. Cellattachment and cell spreading on the difference coating surfaces weremonitored by measuring impedance on the cell-substrate monitoringsystem. Impedance was monitored in real time every 3 minutes for 3hours. The cell index for each time point was calculated by theimpedance monitoring system and plotted as a function of time.

FIG. 9 shows the results of an experiment monitoring morphologicalchanges in Cos-7 cells in response to stimulation with epidermal growthfactor (EGF). Cells were seeded in wells of a 16 well monitoring deviceof the present invention that engaged a device station of acell-substrate monitoring system. The device station was positioned inan incubator held at 37 degrees C. and 5% CO₂. The cells were serumstarved for 8 hours and then stimulated with 50 nanograms/mL of EGF.Control cells did not receive EGF. Impedance was monitored at 3 minuteintervals for 2 hours and then at 1 hour intervals for 14 hours. Thecell index was calculated by the system and plotted as a function oftime. An initial jump in cell index is seen in EGF-treated cells due tomembrane ruffling and actin dynamics in response to EGF. The arrowindicated the point of EGF addition.

D.1. Cell-Based Assays to Test the Effects of Compounds on Cells

In yet another aspect, the present invention provides a method forperforming a cell-based assay investigating the effect of one or moretest compounds on cells, comprising: a) providing a cell-substrateimpedance monitoring system of the present invention; b) introducingcells into at least one of the well of the device that comprises anelectrode array; c) adding at least one test compound to one or more ofthe wells comprising cells and an electrode array; and d) monitoringcell-substrate impedance of the one or more wells before and afteradding the compound, in which changes in impedance can provideinformation about cell responses to the one or more compounds.

Information about cell responses to the one or more compounds includes,but is not limited to, information about cell attachment or adhesionstatus (e.g. the degree of cell spread, the attachment area of a cell,the degree of tightness of cell attachment, cell morphology) on thesubstrate including on the electrodes, cell growth or proliferationstatus; number of viable cells and/or dead cells in the well;cytoskeleton change and re-organization and number of cells goingthrough apoptosis and/or necrosis. Information about cell status mayalso include any compound-cell interaction leading to any change to oneor more of above cell status indicators. For example, if the compoundbinds to a receptor on the cell surface and such binding leads to achange in cell morphology, then the binding of compound to the receptorcan be assayed by the monitored cell-substrate impedance. The cell-basedassays that be performed with above methods include, but not limited to,cell adhesion, cell apoptosis, cell differentiation, cell proliferation,cell survival, cytotoxicity, cell morphology detection, cellquantification, cell quality control, time-dependent cytotoxicityprofiling, IgE-mediated cell activation or stimulation, receptor-ligandbinding, viral and bacterial toxin mediated cell pathologic changes andcell death, detection and quantification of neutralizing antibodies,specific T-cell mediated cytotoxic effect, cell-based assay forscreening and measuring ligand-receptor binding.

The cells used in the assay can be primary cells isolated from anyspecies or can be cells of cell lines. The cells can be geneticallyengineered cells (For example, cells from a genetically modifiedorganism, such as for example from a “gene knockout” organism, or cellsthat have been engineered to overexpress an endogenous gene or atransgene, or cells whose normal gene expression has been manipulated byuse of antisense molecules or silencing RNA.) In some embodiments,different cell types are added to different wells and the behavior ofthe different cell types in response to one or more compounds iscompared.

A test compound can be any compound, including a small molecule, a largemolecule, a molecular complex, an organic molecule, an inorganicmolecule, a biomolecule such as but not limited to a lipid, a steroid, acarbohydrate, a fatty acid, an amino acid, a peptide, a protein, anucleic acid, or any combination of these. A test compound can be asynthetic compound, a naturally occurring compound, a derivative of anaturally-occurring compound, etc.

In preferred methods of the present invention, cells are added to atleast two wells of the cell-substrate impedance monitoring device thatcomprise an electrode array, and at least one well that comprises anelectrode array and comprises cells does not receive a test compound. Acontrol well that does not receive a test compound can be monitored, andits impedance data can be compared with that of wells that do receivecompound to determine the effect of the one or more test compounds oncells.

Impedance can be monitored at regular or irregular time intervals.Preferably, impedance is monitored at three or more time points, atleast one of which is prior to the addition of one or more testcompounds. In one embodiment of the above cell-based assay, thecell-substrate impedance is monitored at least one time point prior toaddition of the test compound, and at regular time intervals thereafter.For example, impedance can be measured at one or more intervals beforeadding the compound and at a regular 2 hour, 1 hour, 30 min or 15 mintime intervals after adding the compound. Preferably, impedance ismeasured at three or more time points spaced at regular intervals. Inthe present application, a real-time assay means allows one to performthe measurement on cell-substrate impedance with various timeresolutions, for example, measurement taking place at a longer timeinterval such as every hour or every two hours, or at a shorter timeinterval every minute or a few minutes.

Impedance can be monitored at one frequency or at more than onefrequency. For example, in some preferred embodiments, impedance ismonitored over a range of frequencies for each time point at whichimpedance is monitored. Preferably, impedance is monitored at least onefrequency between about 1 Hz and about 100 MHz, more preferably at leastone frequency between about 100 Hz and about 2 MHz.

Preferably, data from impedance monitoring of a well that comprisescells and a test compound is compared with data from impedancemonitoring of a well that comprises cells in the absence of a testcompound, however, this is not a requirement of the present invention.For example, it is also possible to compare impedance measurements fromone or more time points prior to the addition of compound to compareimpedance measurements from one or more time points after the additionof compound. Such comparisons can be used directly to assess the cells'response to a compound. It is also possible to calculate a cell index(or cell number index) using the impedance values obtained. Methods ofcalculating a cell index (cell number index) are disclosed herein aswell as in parent application U.S. patent application Ser. No.10/705,447, herein incorporated by reference for disclosures relating tocell number index and its calculation. The cell index calculated fromimpedance measurements of wells receiving compound can be compared withthe cell index calculated from impedance measurements of control wellsto assess the effect of a compound on cells. Alternatively, cell indexcalculated from impedance measurements of wells from one or more timepoints after the addition of a compound can be compared with the cellindex calculated from impedance measurements of wells from one or moretime points prior to the addition of a compound to assess the effect ofa compound on cells. In some preferred embodiments, the cell index canbe used as an indicator of cytotoxicity.

The present invention includes assays in which different concentrationsof a compound are added to wells of a device. The method includes: a)providing a cell-substrate impedance monitoring system of the presentinvention; b) introducing cells into at least two wells of the devicethat each comprise an electrode array; c) adding to at least one well ofthe device comprising cells and an electrode array a first concentrationof a test compound; d) adding to at least one other well of the devicecomprising cells and an electrode array a second concentration of a testcompound; and e) monitoring cell-substrate impedance of the two or morewells before and after adding the compound, in which changes inimpedance can provide information about cell responses to the compound.

Information about cell responses to the compound includes, but is notlimited to, information about cell attachment or adhesion status (e.g.the degree of cell spread, the attachment area of a cell, the degree oftightness of cell attachment, cell morphology) on the substrateincluding on the electrodes, cell growth or proliferation status; numberof viable cells and/or dead cells in the well; cytoskeleton change andre-organization and number of cells going through apoptosis and/ornecrosis. Information about cell status may also include anycompound-cell interaction leading to any change to one or more of abovecell status indicators. For example, if the compound binds to a receptoron the cell surface and such binding leads to a change in cellmorphology, then the binding of compound to the receptor can be assayedby the monitored cell-substrate impedance. The cell-based assays that beperformed with above methods include, but not limited to, cell adhesion,cell apoptosis, cell differentiation, cell proliferation, cell survival,cytotoxicity, cell morphology detection, cell quantification, cellquality control, time-dependent cytotoxicity profiling, IgE-mediatedcell activation or stimulation, receptor-ligand binding, viral andbacterial toxin mediated cell pathologic changes and cell death,detection and quantification of neutralizing antibodies, specific T-cellmediated cytotoxic effect, cell-based assay for screening and measuringligand-receptor binding.

The cells and test compound used in the assay can be as described abovefor assays testing effects of test compounds. In preferred methods ofthe present invention, cells are introduced into at least three wells ofthe device that each comprise an electrode array, and at least one wellthat comprises an electrode array and comprises cells does not receivetest compound. A control well that does not receive a test compound canbe monitored, and its impedance data can be compared with that of wellsthat do receive compound to determine the effect of the one or more testcompounds on cells.

Impedance monitoring can be as described immediately above for assaystesting effects of test compounds.

Preferably, data from impedance monitoring of wells that comprise cellsand different concentrations of test compounds are compared. Suchcomparisons can be used directly to assess the cells' response toincreasing concentrations of a compound. It is also possible tocalculate a cell index (or cell number index) using the impedance valuesobtained. Methods of calculating a cell index (cell number index) aredisclosed herein as well as in parent application U.S. application Ser.No. 10/705,447, herein incorporated by reference for disclosure of cellindex number and method of calculating cell index number. In somepreferred embodiments, the cell index can be used as an indicator ofcytotoxicity.

The cell index calculated from impedance measurements of wells receivingdifferent concentrations of compound can be compared to assess theeffect of a compound on cells. Alternatively, cell index calculated fromimpedance measurements of wells comprising different concentrations of acompound can be compared. Dose response relationships can be derivedfrom such comparisons. In some preferred embodiments, time dependentIC50 values may be calculated from cell index values for compounds thatexhibit cytotoxicity or inhibit particular cell responses.

In one embodiment of the method, analyzing the cytotoxicity response mayinclude derivation of the slope of change in the time dependentcytotoxicity response at a given compound concentration. In yet anotherembodiment of the method, analyzing real-time cytotoxicity response mayinclude derivation of high-order derivatives of the time dependentcytotoxicity response with respect to time at a given compoundconcentration.

Cell-Based Assays with More Than One Compound

In yet another aspect, the present invention provides a method forperforming a cell-based assay investigating the effect of two or moretest compounds on cells. The method includes: a) providing acell-substrate impedance monitoring system of the present invention; b)introducing cells into at least two wells of the device that eachcomprise an electrode array; c) adding to at least one well of thedevice comprising cells and an electrode array a first test compound; d)adding to at least one other well of the device comprising cells and anelectrode array a second test compound; and e) monitoring cell-substrateimpedance of at least one well comprising cells and a first compound andat least one well comprising cells and a second compound, in whichchanges in impedance can provide information about cell responses to thefirst and second compounds.

Preferably, time-dependent responses of cells to the first compound andthe second compound are compared to see how similar or different theresponses from the two compounds are. In one preferred embodiment ofthis method, time-dependent cytotoxic responses are compared.

The cells and test compound used in the assay can be as described abovefor assays testing effects of test compounds. In preferred methods ofthe present invention, cells are introduced into at least three wells ofthe device that each comprise an electrode array, and at least one wellthat comprises an electrode array and comprises cells does not receive atest compound. A control well that does not receive a test compound canbe monitored, and its impedance data can be compared with that of wellsthat receive a compound to determine the effect of the test compounds oncells.

Impedance monitoring can be as described immediately above for assaystesting effects of test compounds.

Preferably, data from impedance monitoring of wells that comprisedifferent test compounds are compared. In one preferred embodimentimpedance monitoring is performed for the first compound at multipledose concentrations. In another embodiment, time-dependent cellularresponses are determined for the second compound at multiple doseconcentrations. In yet another embodiment, time-dependent cellularresponses are determined for both first compound and second compound atmultiple dose concentrations.

In another embodiment of above method, the first compound is a compoundwith a known mechanism for its cytotoxic effect and the second compoundis a compound with an unknown mechanism for its cytotoxic effect. If thetime dependent cytotoxic responses from the second compound are similarto that of the first one, the second compound may follow a similarmechanism for its cytotoxic effect to the first compound.

Various approaches may be used in comparing the cytotoxic responses ofthe compounds. A cell index (or cell number index) can optionally becalculated using the impedance values obtained. In one embodiment of themethod described above, time dependent IC50 may be derived for thecompounds and comparison between their cytotoxic responses is done bycomparing their time dependent IC50 curves based on cell index values.If the IC50 curves follow a similar time-dependent trend, the twocompounds may follow a similar mechanism for inducing cytotoxicityeffects. In another embodiment of the method described, directcomparison of time-dependent cytotoxic responses of two compounds aredone where the concentrations for the two compounds may be the same ormay be different. Direct comparison between time-dependent cytotoxicresponses may be done by analyzing the slope of change in the measuredresponses (that is equivalent to the first order derivative of theresponse with respect to time) and comparing the time-dependent slopesfor the two compounds. In another approach, the time-dependent cytotoxicresponses may be analyzed for their higher order derivatives withrespect to time. Comparing such high order derivatives may provideadditional information as for the mechanisms of compound-inducedcytotoxicity.

Cell-Based Assays with More Than One Cell Type

In yet another aspect, the present invention provides a method forcytotoxicity profiling for a compound on multiple cell types,comprising: a) providing a cell-substrate impedance monitoring system ofthe present invention; b) introducing a first type of cells into atleast one well of the device that comprises an electrode array; c)introducing a second type of cells into at least one other well of thedevice that comprises an electrode array; d) adding a test compound toat least one well comprising cells of a first type and to at least onewell comprising cells of a second type; and e) monitoring cell-substrateimpedance of at least one well comprising cells of a first type and testcompound and of at least one well comprising cells of a second type andtest compound, in which changes in impedance can provide informationabout cell responses to the first and second compounds.

Preferably, time-dependent responses of the first and second types ofcells are compared to see how similar or different the responses fromthe two types of cells are. In one preferred embodiment of this method,time-dependent cytotoxic responses are compared.

The cell types used in the assay can be primary cells isolated from anyspecies or can be cells of cell lines. In some preferred embodiments,the different cell types are the same type of cell from differentindividuals, and thus have different genotypes. One or more of the celltypes can be genetically engineered (For example, cells from agenetically modified organism, such as for example from a “geneknockout” organism, or cells that have been engineered to overexpress anendogenous gene or a transgene, or cells whose normal gene expressionhas been manipulated by use of antisense molecules or silencing RNA.) Inthese cases, genetically modified cells can be compared with controlcells. In some embodiments, three or more different cell types are addedto different wells and the behavior of the three or more different celltypes in response to one or more compounds is compared.

The test compound or compounds used in the assay can be as describedabove for assays testing effects of test compounds. In preferred methodsof the present invention, cells are introduced into at least three wellsof the device that each comprise an electrode array, and at least onewell that comprises an electrode array and comprises cells does notreceive a test compound. A control well that does not receive a testcompound can be monitored, and its impedance data can be compared withthat of wells that receive a compound to determine the effect of thetest compounds on cells. In preferred embodiments of the presentinvention, for each cell type tested there is a control performed inwhich the control does not receive test compound.

Impedance monitoring can be as described immediately above for assaystesting effects of test compounds.

Preferably, data from impedance monitoring of wells that comprisedifferent cell types are compared. In one preferred embodiment impedancemonitoring is performed for different cell types exposed to multipledose concentrations of a compound. In some embodiments, multiplecompounds can be tested with multiple cell types. In some embodiments,multiple compounds at multiple concentrations can be tested withmultiple cell types.

In one embodiment of the method, analyzing real-time cytotoxicityresponse may include the derivation of time-dependent IC50 values forthe compound on the multiple cell types. In another embodiment of themethod, analyzing real-time cytotoxicity response may include derivationof the slope of change in the time dependent cytotoxicity response at agiven compound concentration. In yet another embodiment of the method,analyzing real-time cytotoxicity response may include derivation ofhigh-order derivatives of the time dependent cytotoxicity response withrespect to time at a given compound concentration.

In one embodiment of the method, analyzing real-time cytotoxicityresponses may include the derivation of time-dependent IC50 values forthe compound on the multiple cell types. In yet another embodiment, theabove methods are applied to perform cytotoxicity profiling of multiplecompounds on multiple cell types.

In another embodiment of the method, analyzing real-time cytotoxicityresponse may include derivation of the slope of change in the timedependent cytotoxicity response at a given compound concentration. Inyet another embodiment of the method, analyzing real-time cytotoxicityresponse may include derivation of high-order derivatives of the timedependent cytotoxicity response with respect to time at a given compoundconcentration.

Some examples of compound assays that can be performed using acell-substrate impedance system of the present invention are provided byway of illustration with reference to the figures. In these examples,cell index is calculated using the same method as the Cell Indexcalculation method (A) as described in Section C of the presentapplication. In some of the figures of the present application,Normalized Cell Index was plotted. The Normalized Cell Index at a giventime point is calculated by dividing the Cell Index at the time point bythe Cell Index at a reference time point. Thus, the Normalized CellIndex is 1 at the reference time point.

As described in the present application, if the cell attachmentconditions remain unchanged or exhibit little change over the course ofan assay that uses impedance monitoring, then the larger the cell index,the larger the number of the cells in the wells. A decrease in cellindex suggests that some cells are detaching from the substrate surfaceor dying under the influence of the compound. An increase in cell indexsuggests that more cells are attaching to the substrate surfaces,indicating an increase in overall cell number.

FIG. 10 shows curves that represent the time-dependent cell index forH460 cells treated with different concentrations of the anticancer drugpaclitaxel. In this experiment, H460 cells were introduced into wells ofa 16× cell-substrate impedance monitoring device. The device waspositioned on a device station that was located in an incubatormaintaining conditions of 37 degrees C. and 5% CO₂. The cells werecultured and treated at their exponential growth phase with differentconcentrations of paclitaxel. The dynamic response of the cells todifferent doses of paclitaxel was monitored by monitoring cell-substrateimpedance in real time every 15 minutes for 50 hours after treatmentusing a cell-substrate impedance monitoring system. The cell-substrateimpedance monitoring system calculated the cell index at each time pointmonitored and plotted the cell index as a function of time. Forpaclitaxel concentrations between 67 nanomolar and 500 nanomolar, H460cells exhibited a gradual decrease in cell index after compoundaddition. However, the cell index reached a minimum at a time dependenton the compound concentration, between about 15 hours and 20 hours aftercompound addition. After that point, there was a gradual increase incell index in these wells. The cell index for compound concentration of33 nanomolar exhibited a near-constant value for up to about 15 hoursafter compound addition. After 15 hours following compound addition, thecell index exhibited a gradual increase.

FIG. 11 shows curves that represent the time-dependent cell index forH460 cells treated with anticancer drug AC101103. H460 cells wereintroduced into wells of a 16× cell-substrate impedance monitoringdevice. The device was positioned on a device station that was locatedin an incubator maintaining conditions of 37 degrees C. and 5% CO₂. Thecells were cultured and treated at their exponential growth phase withdifferent concentrations of AC101103. The dynamic response of the cellsto different doses of AC101103 was monitored by measuring impedance inreal time every 30 minutes for about 20 hours after treatment on thecell-substrate monitoring system.

Notably, the time-dependent cell index in FIG. 11 is significantlydifferent from those shown in FIG. 10. For compound concentrations at3.125 microgram/ml, 6.25 microgram/ml and 12.5 microgram/ml, the cellindex exhibited a near-constant value for about 5 hrs, about 15 hrsand >20 hrs respectively. For compound concentrations at 3.125microgram/ml and 6.25 microgram/ml, the cell index started to increaseafter about 5 hrs and about 15 hrs following compound addition. For thecompound concentration of 25 microgram/ml, there was a gradual, yet slowdecrease in the cell index after compound addition. For the compoundconcentration of 50 microgram/ml, there was an about 10 hr time periodover which the cell index remained near-constant, and after that, thecell index decreased steadily.

FIG. 12 shows dynamic drug response curves of A549 cells treated withdoxorubicin. 10,000 A549 cells were seeded into each well of a 16×device. The device was positioned on a device station that was locatedin an incubator maintaining conditions of 37 degrees C. and 5% CO₂. Cellattachment and cell growth were monitored on a cell-substrate impedancesystem in real time before treatment by monitoring impedance at regularintervals. When the cells were in exponential growth phase, doxorubicinat different concentrations was added to the wells. The same volume ofthe solvent used to dissolve the drug was added to some wells as acontrol. The time, and drug dose dependent cell response (calculated ascell index) to doxorubicin was recorded in real time on thecell-substrate impedance monitoring system as shown in this figure.

E. Cell-Substrate Impedance Assays to Monitor IgE-Mediated CellActivation

The present invention also includes methods of monitoring cell-substrateimpedance of cells stimulated by IgE. The method is based on quantifyingin real time the cytoskeletal changes that result from antigen bindingto the IgE-Fc(epsilon)RI complex on the surface of responsive cells,such as, but not limited to, mast cells. The electronic assays providedrely on cytoskeletal dynamics that are an intrinsic mast cell responseto antigen and an essential part of the mast cell activation program,and precludes the need for establishing reporter cell lines or usingother assay reagents. Furthermore, since the assay is performed in realtime, both antigen-dependent and antigen-independent response toIgE-mediated activation of mast cells can be monitored in the sameassay.

The assays described herein that monitor cell-substrate impedance ofcells stimulated by IgE can use any cell-substrate impedance measuringdevice, including but not limited to those described in parent U.S.patent application Ser. No. 10/705,447 and herein. A cell-substrateimpedance device useful in the methods of the present invention refersto any device that has a surface suitable for cells and has electrodesthat cells can settle on and interact with. The measurement on theelectrode impedance can reflect the cell status such as cell number,cell morphology, or cell adhesion. In preferred embodiments, acell-substrate impedance device useful in the methods of the presentinvention comprises a substrate that comprises one or more electrodearrays on its surface, in which each of the one or more electrode arrayscomprises two electrodes or electrode structures, where the twoelectrodes or electrode structures have substantially the same surfacearea. In operation, a cell-substrate impedance device used in themethods of the present invention can detect impedance changes at one ormore frequencies due to changes in cell number, cell size, cellmorphology, cell attachment to the substrate, and the quality of cellattachment to the substrate.

Preferably a cell-substrate impedance device used in the methods of thepresent invention comprises at least one fluid container that surroundsan electrode array of the device and provides a fluid-impermeablecontainer for cells being monitored. In preferred embodiments, a devicecomprises at least two arrays and at least two receptacles in the formof wells, where each array of the device is encompasses by a well. Morepreferably, a device used in the screening methods described in thepresent application comprises at least 8 wells (for example, devicescomprising 16 or 96 wells), of which the majority comprise electrodearrays, so that assays can be performed in a high-throughput fashion.Cells can optionally be assayed in multiple multi-well devicessimultaneously (for example, connected to the same impedance analyzer,engaged with a device station that can engage more than one multi-welldevice, or connected to separate impedance analyzers) to increasehigh-throughput capacity.

Systems for monitoring cell-substrate impedance that comprise one ormore multiwell devices, an impedance analyzer, and a device station aredescribed herein and are preferred but not required for use in themethods of the present invention.

The cells used in the IgE stimulation assays can be any cells, isolatedfrom one or more organisms or from cell lines. Cells can be isolatedfrom blood or other tissues of one or more organisms. Preferably thecells are mammalian cells. Preferably the cells are mast cells,basophils, or eosinophils, but that is not a requirement of the presentinvention. The cells can be genetically engineered cells of any typethat express components of the IgE response pathway. Cells can beengineered to inappropriately express or overexpress components of theIgE response pathway. Cells can also be engineered to express dominantnegative or other altered versions of components of the IgE responsepathway, or to ablate expression of components of the IgE responsepathway (for example, by use of homologous recombination knockouts,antisense or silencing RNA technologies). As used herein, “components ofthe IgE response pathway” also includes molecules suspected of beingcomponents of the IgE response pathway.

In some embodiments of some aspects of the present invention, RBL-2H3rat mast cells can be used investigate the IgE-mediated signalingmechanism and also to assess the effect of various inhibitors on thesignaling pathways leading to mast cell degranulation and mediatorrelease. RBL-2H3 cells offer the advantage of being maintained andexpanded in culture and a large body of literature which has utilizedthis cell line as a model system for IgE-mediated mast cell activation.However, the methods and systems of the present invention can readily beadapted to other cells that may be of academic and pharmaceuticalinterest. The cells include but are not limited to mouse bone marrowmast cells, human lung mast cells, human skin mast cells, and mast cellsand basophils from other mammalian species.

The following subsections disclose assays that use impedance monitoringdevices that can measure both IgE binding and antigen-mediatedIgE-Fc(epsilon)RI cross-linking leading to mast cell morphologicalchanges and degranulation. The assays can be performed inhigh-throughput format, in real time and without the need for any otherreagents or cellular manipulation.

E.1. Methods for Monitoring Changes in Cell-Substrate Impedance inResponse to IgE Stimulation

IgE-mediated cell stimulation has been shown to lead to dramaticmorphological changes in cells such as mast cells. For example, RBL-2H3mast cells are transformed from spindle shape morphology to flattenedand fibroblastic cell morphology. Cell-substrate impedance monitoringdevices of the present invention can be used to detect cell shapechanges and changes in cell-substratum interaction that result fromIgE-mediated stimulation of responsive cells.

The method comprises: a) providing a device for cell-substrate impedancemonitoring that comprising at least one well that comprises at least oneelectrode array; b) connecting an impedance analyzer to the device; c)introducing cells into one or more wells of the device that comprise anelectrode array; d) adding IgE to the one or more wells comprisingcells; e) adding at least one antigen, at least one allergen, or atleast one IgE crosslinker to the one or more wells comprising cells; ande) monitoring cell-substrate impedance of the one or more wellscomprising cells.

In the methods of the present invention, a device for cell-substrateimpedance monitoring is a device that comprises a substrate having oneor more electrode arrays on its surface each of which is encompassed bya fluid container in the form of a well. Each of the one or moreelectrode arrays comprises two electrodes or electrode structures, wherethe two electrodes or electrode structures have substantially the samesurface area, in which the device, when connected to an impedanceanalyzer, can detect impedance changes at one or more frequencies due tochanges in cell number, cell size, cell morphology, cell attachment tothe substrate, or the quality of cell attachment to the substrate.

The cells can be any cells whose response or possible response to IgEstimulation is of interest. Preferred cells are mast cells, eosinophils,basophils, or genetically engineered cells. In some preferredembodiments, the cells are of mammalian origin.

Ig E can be added before, after, or simultaneous with the addition ofthe antigen, allergen, or IgE crosslinker. IgE can be added to wells ata concentration of from about 10 nanograms per milliliter to about 1microgram per milliliter. Preferably, one well of the device thatcomprises an electrode array and cells does not receive IgE to provideat least one control well, but this is not a requirement of the presentinvention.

Antigens, allergens, or crosslinkers can be known or suspected antigens,allergens, or crosslinkers. The concentration added to the wells can befrom about 1 nanogram per milliliter to about 1 microgram permilliliter. Different wells may receive different concentrations of anantigen, allergen, or crosslinker. Or, for replica purposes, multiplewells may receive same concentrations of an antigen, allergen orcrosslinker. Preferably, one well of the device that comprises anelectrode array and cells does not receive antigen, allergen, orcrosslinker to provide at least one control well, but this is not arequirement of the present invention.

The method can be used to assess and quantify the morphological changesthat occur in mast cells as a result of IgE stimulation and IgEcross-linking with an antigen by using cell-substrate impedancetechnology. Impedance is preferably monitored during two or more phasesof the experiment. For example, in one embodiment, impedance ismonitored before and after the addition of IgE to the wells. In anotherembodiment, impedance is monitored before and after the addition ofantigen, allergen, or crosslinker to the wells. In yet anotherembodiment, impedance can be monitored before and after the addition ofIgE plus antigen, allergen, or crosslinker to the wells. Impedance canbe also be monitored after the addition of IgE to the wells and beforethe addition of antigen, allergen, or crosslinker to the wells, as wellas after the addition of antigen, allergen, or crosslinker to the wells.In some preferred embodiments, impedance is monitored before addition ofIgE to the wells, after the addition of IgE to the wells and before theaddition of antigen, allergen, or crosslinker to the wells, and afterthe addition of antigen, allergen, or crosslinker to the wells.

Impedance can be monitored at two or more time points. Preferably,impedance is monitored at least three time points. Preferably, impedanceis monitored at least three time points at two or more phases of theassay. Impedance at each time point can be monitored at one or at morethan one frequency. Data obtained from impedance monitoring can be usedto assess morphological changes to cells that result from IgEstimulation. For example, impedance before and after addition of IgE,and/or before and after addition of antigen, allergen, or crosslinkercan be compared for each well. Impedance data can be compared for wellsthat receive IgE and control wells that do not receive IgE. Impedancedata can be compared for wells that receive antigen, allergen, orcrosslinker and control wells that do not receive antigen, allergen, orcrosslinker. Impedance data can be used to calculate a cell index, whichcan be used for comparisons.

One exemplary protocol is as follows:

-   -   (1) Add a predetermined number of mast cells to the wells of        either a 16-well or 96 well cell-substrate impedance measurement        device that is attached to an impedance analyzer.    -   (2) Allow the cells to attach and grow for 16-20 hours while        recording the IgE anywhere from 10 nanogram/mL to 1 microg/mL        final concentration and continue recording the cellular response        using a cell-substrate impedance monitoring system as described        herein.    -   (3) At 16-20 hours after IgE stimulation, change media to serum        free media. Allow the cells to recover for 30 minutes and then        stimulate with antigen. Continue monitoring the cellular        response to antigen on the impedance monitoring system.        E.2. Methods of Screening for Compounds That can Modulate a        Cellular Response to IgE Stimulation

The present invention also includes methods of assessing the effect ofone or more compounds on one or more cellular responses to IgE-mediatedstimulation. The methods comprise: a) providing a device forcell-substrate impedance monitoring that comprising at least one wellthat comprises an electrode array; b) connecting an impedance analyzerto the device; c) introducing cells into one or more wells of the devicethat comprise an electrode array; d) adding at least one test compoundto one or more of the one or more wells that comprise cells; e) addingIgE to the one or more wells comprising cells and at least one testcompound; e) adding at least one antigen, at least one allergen, or atleast one IgE crosslinker to the one or more wells comprising cells andat least one test compound; and e) monitoring cell-substrate impedanceof the one or more wells comprising cells and at least one testcompound.

In the methods of the present invention, a device for cell-substrateimpedance monitoring is a device that comprises a substrate having oneor more electrode arrays on its surface, each of which is encompassed bya fluid container in the form of a well. Each of the one or moreelectrode arrays comprises two electrodes or electrode structures, wherethe two electrodes or electrode structures have substantially the samesurface area. The device, when connected to an impedance analyzer, candetect impedance changes at one or more frequencies due to changes incell number, cell size, cell morphology, cell attachment to thesubstrate, or the quality of cell attachment to the substrate.

Preferably, a device used in the methods of the present invention ispart of a cell-substrate impedance monitoring system as describedherein.

The cells can be any cells that have a detectable response to IgEstimulation. Preferred cells are mast cells, eosinophils, basophils, orgenetically engineered cells. In preferred embodiments, the cells are ofmammalian origin.

Ig E can be added before, after, or simultaneous with the addition ofthe antigen, allergen, or IgE crosslinker. IgE can be added to wells ata concentration of from about 10 nanograms per milliliter to about 1microgram per milliliter. Optionally, one well of the device thatcomprises an electrode array and cells does not receive IgE to provide acontrol well.

Antigens can be known or suspected antigens, allergens, or crosslinkers.The concentration added to the wells can be from about 1 nanogram permilliliter to about 1 microgram per milliliter. Different wells mayreceive different concentrations of an antigen, allergen, orcrosslinker. Or, for replica purposes, multiple wells may receive sameconcentrations of an antigen, allergen or crosslinker. Optionally, onewell of the device that comprises an electrode array and cells does notreceive antigen, allergen, or crosslinker to provide a control well.

A test compound can be any compound, including a small molecule, a largemolecule, a molecular complex, an organic molecule, an inorganicmolecule, a biomolecule such as but not limited to a lipid, a steroid, acarbohydrate, a fatty acid, an amino acid, a peptide, a protein, anucleic acid, or any combination of theses. A test compound can be asynthetic compound, a naturally occurring compound, a derivative of anaturally-occurring compound, etc. The structure of a test compound canbe known or unknown. One or many compounds can be assayed in a singleexperiment. Compounds can be tested in combination. Differentconcentrations of a given compound can be assayed using the methods ofthe present invention.

The advent of combinatorial chemistry has allowed for the generation oflarge and diverse compound libraries that can be used to screen forpotential inhibitors of IgE-mediated mast cell activation. Assays can beperformed in cell-substrate impedance monitoring systems of the presentinvention to screen compound libraries for potential inhibitors inhigh-throughput manner.

Test compounds can be compounds that interfere with IgE-mediatedsignaling through the Fc(epsilon)RI receptor expressed on the surface ofmast cells and basophils. For example, a test compound can be anantibody raised against the IgE. or a recombinant protein comprising allor a portion of the antigen binding domain of Fc(epsilon)RI that can betested for its ability to specifically bind to the IgE and prevent itfrom interacting with the endogenous Fc(epsilon)RI. Test compounds canalso be candidates for small molecule inhibitor compounds that can bindto the IgE-binding pocket of Fc(epsilon)RI and block its interactionwith the IgE molecule. Test compounds can also be siRNAs that can betested for their ability to down-regulate the expression ofFc(epsilon)RI on the surface of mast cells.

Test compounds can also be screened to identify compounds that can actintracellularly to inhibit cellular response to IgE stimulation. Forexample, test compounds can be screened to identify inhibitors ofkinases or lipases that have a role in cell signaling in response to IgEstimulation. For example, the methods of the present invention can beused to screen for compounds that can inhibit protein kinase C, SRC,Syk, or PLC(gamma).

A test compound is preferably added to wells prior to the addition ofIgE, but can also be added after the addition of IgE and before theaddition of antigen, or after the addition of IgE and antigen. Inembodiments where IgE and antigen are added at the same time, a testcompound can be added after but is preferably added after the additionof IgE and antigen.

The method can be used to assess and quantify the effects of a compoundon the response of cells, such as but not limited to mast cells, as aresult of IgE by using cell-substrate impedance technology. Impedance ispreferably monitored during two or more phases of the experiment.Depending on the experimental protocol, impedance is preferablymonitored after adding IgE to the one or wells, and is preferablymonitored before and after adding IgE to the one or wells. Impedance ispreferably monitored after the addition of test compound, before andafter the addition of IgE. Impedance can also be monitored after theaddition of test compound and IgE, before and after addition antigen. Insome preferred embodiments, impedance is monitored after the addition oftest compound and before addition of IgE to the wells, and after theaddition of IgE to the wells and before the addition of antigen,allergen, or crosslinker to the wells, and after the addition ofantigen, allergen, or crosslinker to the wells.

Impedance is preferably monitored at two or more time points.Preferably, impedance is monitored at least three time points.Preferably, impedance is monitored at least three time points at two ormore phases of the assay. Impedance at each time point can be monitoredat one or at more than one frequency. Data obtained from impedancemonitoring can be used to assess the effects of a test compound on themorphological changes to cells that result from IgE stimulation. Forexample, impedance before and after addition of test compound, and/orbefore and after addition of IgE, and/or before and after addition ofantigen, allergen, or crosslinker can be compared for each well.Impedance data can be compared for wells that receive test compound andcontrol wells that do not receive test compound Impedance data can beused to calculate a cell index, which can be used for comparisons. Suchcomparisons can be used to identify compounds that inhibit the responseof cells such as mast cells to IgE stimulation. For example, IC50s canbe calculated from recorded impedance values for one or more testcompounds that inhibit a cellular response to IgE stimulation

One exemplary protocol for compound screening is as follows:

-   -   (1) A predetermined number of mast cells is added to the wells        of either a 16-well or 96 well cell-substrate impedance        measurement device that is part of a cell impedance monitoring        system.    -   102. The attachment and growth of the cells is monitored using        the impedance monitoring system.    -   103. The cells are pre-incubated with increasing concentrations        of one or more potential inhibitors of interest (anti-IgE        antibody, recombinant fragment of the Fc(epsilon)RI antigen        binding domain or small molecular compounds) for a defined        length of time.    -   104. IgE specific for the cell line used is administered and the        transient morphological changes of the cells due IgE stimulation        is electronically monitored by the system. The extent of        inhibition and IC-50 determination is assessed by comparing the        peak response of the recording of the cells in the presence of        increasing concentrations of the inhibitor.

In another example of the methods of the present invention, mast cellsor other cells of interest are dispensed into a 96 well device either byliquid handling station or by multi-channel pipette. Cell attachment andgrowth is monitored as above. To assess the effect of the inhibitors onIgE-alone mediated signaling, inhibitors at a single concentration ormultiple concentrations are pre-incubated with the cells prior to IgEapplication. The cellular response to IgE in the presence of the drug isthen monitored as described above. Alternatively, if the effect of thedrugs are to be assessed in response to the antigen cross-linking of theIgE-Fc(epsilon)RI complex, the inhibitors are preincubated with thecells just prior to the addition of the antigen as described above.

E.3 Methods for Target Validation of Enzymes and Proteins Involved inthe Signaling Pathway Initiated by Engagement of the High-AffinityFc(epsilon)RI Receptor in the Presence or Absence of IgE Cross-Linkingby Antigen Using the Cell-Substrate Impedance Technology

The intracellular signaling pathway that is stimulated by engagement ofthe Fc(epsilon)RI by IgE involves the activation of key enzymes such asbut not limited to kinases, phosphatases and phospholipases. Thesedownstream mediators of the cellular response to IgE are potentialtargets for pharmaceutical drug discovery. However, prior to screeningfor potential inhibitors of these target proteins and enzymes, they mustbe validated to ascertain that they can interfere with IgE-mediatedsignaling. This can be achieved by introducing into responsive cells bytransfection, electroporation or viral infection the DNA encoding forthe dominant negative versions of suspected target proteins or one ormore siRNAs that target and reduce the expression of these proteins.Alternatively, antisense reagents can be used to reduce or ablateexpression of a suspected target.

The method includes: a) providing a device for cell-substrate impedancemonitoring that comprising at least one well that comprises an electrodearray; b) connecting an impedance analyzer to the device; c) introducingcells into one or more wells of the device that comprise an electrodearray, wherein said cells are genetically modified to alter the functionof, or reduce or ablate the expression of, a suspected target molecule;d) adding IgE to the one or more wells comprising cells; e) adding atleast one antigen, at least one allergen, or at least one IgEcrosslinker to the one or more wells comprising cells; and e) monitoringcell-substrate impedance of the one or more wells comprising cells.

In the methods of the present invention, a device for cell-substrateimpedance monitoring is a device that comprises a substrate having oneor more electrode arrays on its surface, each of which is encompassed bya fluid container in the form of a well. Each of the one or moreelectrode arrays comprises two electrodes or electrode structures, wherethe two electrodes or electrode structures have substantially the samesurface area. The device, when connected to an impedance analyzer, candetect impedance changes at one or more frequencies due to changes incell number, cell size, cell morphology, cell attachment to thesubstrate, or the quality of cell attachment to the substrate.

Devices for measuring cell-substrate impedance are described in parentU.S. patent application Ser. No. 10/705,447 and in the presentapplication. Preferably a cell-substrate impedance device used in themethods of the present invention comprises at least one fluid containerthat surrounds an electrode array of the device and provides afluid-impermeable container for cells being monitored. In preferredembodiments, a device comprises at least two arrays and at least tworeceptacles in the form of wells, where each array of the device isencompassed by a well. More preferably, a device used in the screeningmethods described in the present application comprises at least 8 wells(for example, devices comprising 16 or 96 wells), of which the majoritycomprise electrode arrays, so that assays can be performed in ahigh-throughput fashion. Cells can optionally be assayed in multiplemulti-well devices simultaneously (for example, connected to the sameimpedance analyzer, engaged with a device station that can engage morethan one multi-well device, or connected to separate impedanceanalyzers) to increase high-throughput capacity.

Systems for monitoring cell-substrate impedance that comprise one ormore multiwell devices, an impedance analyzer, and a device station aredescribed herein and are preferred but not required for use in themethods of the present invention.

The genetically modified cells can be any type of cells that isresponsive to IgE stimulation. Preferably, mast cells are used, such asRBL-2H3 rat mast cells. Methods of genetically modifying cells are wellknown in the art, and include introducing expression constructs thatdirect the expression of altered versions of the suspected targetprotein (for example, dominant negative versions of a protein),introducing expression constructs that direct the expression ofantisense RNAs that can reduce or ablate the expression of a targetprotein, and introducing expression constructs that direct theexpression of silencing RNAs that can reduce of ablate the expression ofa target protein. Antisense or gene silencing reagents can also be addedto cultured cells before or after adding the cells to a device of thepresent invention.

Preferably, control cells that have not been genetically modified arealso assayed alongside genetically modified cells.

Ig E can be added before, after, or simultaneous with the addition ofthe antigen, allergen, or IgE crosslinker. IgE can be added to wells ata concentration of from about 10 nanograms per milliliter to about 1microgram per milliliter. Optionally, one well of the device thatcomprises an electrode array and cells does not receive IgE to provide acontrol well.

Antigens used in the assays can be antigens, allergens, or crosslinkers.The concentration added to the wells can be from about 1 nanogram permilliliter to about 1 microgram per milliliter.

The method can be used to assess and quantify the effects of a compoundon the response of cells, such as but not limited to mast cells, as aresult of IgE by using cell-substrate impedance technology. Impedance ispreferably monitored during two or more phases of the experiment.Depending on the experimental protocol, impedance is preferablymonitored after adding IgE to the one or wells, and is preferablymonitored before and after adding IgE to the one or wells. Impedance ispreferably monitored after the addition of test compound, before andafter the addition of IgE. Impedance can also be monitored after theaddition of test compound and IgE, before and after addition antigen. Insome preferred embodiments, impedance is monitored after the addition oftest compound and before addition of IgE to the wells, and after theaddition of IgE to the wells and before the addition of antigen,allergen, or crosslinker to the wells, and after the addition ofantigen, allergen, or crosslinker to the wells.

Impedance is preferably monitored at two or more time points.Preferably, impedance is monitored at least three time points.Preferably, impedance is monitored at least three time points at two ormore phases of the assay. Impedance at each time point can be monitoredat one or at more than one frequency. For example, impedance valuesbefore and after addition of IgE, and/or before and after addition ofantigen can be compared for each well. Impedance data can be comparedfor wells that comprise genetically manipulated cells and control wellscomprise cells that are not genetically manipulated. Impedance data canbe used to calculate a cell index, which can be used for comparisons.Such comparisons can be used to identify genes that affect the responseof cells to IgE stimulation.

Data obtained from impedance monitoring can be used to assess theeffects of a genetic manipulation (such as expression knockdown,expression knockout, or dominant negative expression) on themorphological changes to cells that result from IgE stimulation.Identification of a genetic manipulation that affects the morphologicalchanges to cells that result from IgE stimulation is used to identify agene, and thus a gene product, that has a role in the IgE response.

One example of an assay of the present invention for validating a targetis as follows:

-   -   (1) Provide cells having either the DNA for the dominant        negative version of a protein of interest or for siRNA targeting        of a protein of interest.    -   (2) Transfer cells to wells of a device of an impedance        monitoring system and monitor the attachment and growth of the        cells as described above. Alternatively, gene interfering        reagents can be directly introduced into the cells in the wells        of the device.    -   (3) Cells are stimulated with IgE in the presence or absence of        antigen and the cellular response is recorded by the impedance        monitoring system as previously discussed. The ability of a        genetic construct or reagent to interfere with the response of        cells to IgE stimulation by antigen will allow identification of        the molecule the construct or reagent acts against as a        potential target for drug discovery.        E.4 Method for Screening Genetic Markers That Determine or        Influence Engagement of High-Affinity Fc(epsilon)RI        Cross-Linking and Subsequently Mast Cell Activation

It is well known that host genetic background determines the types andseverity of allergic response to similar antigens. The present inventionalso includes methods of comparing the responses of cell of differentgenotypes to antigen-mediated IgE stimulation. The responses of cells ofdifferent genotypes to antigen-mediated IgE stimulation can becorrelated with any of a number of genetic markers that the cells ofdifferent genotypes display.

The method includes: a) providing a device for cell-substrate impedancemonitoring that comprising at least one well that comprises an electrodearray; b) connecting an impedance analyzer to the device; c) introducingcells of a first genotype into at least one well of the device; d)introducing cells of a second genotype into at least one other well ofthe device; e) adding IgE to at least one well comprising cells of afirst genotype and at least one well comprising cells of a secondgenotype; e) adding at least one antigen to the one or more wellscomprising cells of a first genotype and to the one or more wellscomprising cells of a second genotype; e) monitoring cell-substrateimpedance of the one or more wells comprising cells of a first genotypeand of the one or more wells comprising cells of a second genotype; andf) comparing the cell-substrate impedance values of the one or morewells comprising cells of a first genotype with the cell-substrateimpedance values of the one or more wells comprising cells of a secondgenotype.

Preferably, the method further includes: correlating at least onegenetic marker with the cell substrate impedance values obtained frommonitoring cells of said first genotype and cells of said secondgenotype.

In the methods of the present invention, a device for cell-substrateimpedance monitoring is a device that comprises a substrate having oneor more electrode arrays on its surface, each of which is encompassed bya fluid container in the form of a well. Each of the one or moreelectrode arrays comprises two electrodes or electrode structures, wherethe two electrodes or electrode structures have substantially the samesurface area. The device, when connected to an impedance analyzer, candetect impedance changes at one or more frequencies due to changes incell number, cell size, cell morphology, cell attachment to thesubstrate, or the quality of cell attachment to the substrate.

Devices for measuring cell-substrate impedance are described in parentU.S. patent application Ser. No. 10/705,447 and in the presentapplication. Preferably a cell-substrate impedance device used in themethods of the present invention comprises at least one fluid containerthat surrounds an electrode array of the device and provides afluid-impermeable container for cells being monitored. In preferredembodiments, a device comprises at least two arrays and at least tworeceptacles in the form of wells, where each array of the device isencompassed by a well. More preferably, a device used in the screeningmethods described in the present application comprises at least 8 wells(for example, devices comprising 16 or 96 wells), of which the majoritycomprise electrode arrays, so that assays can be performed in ahigh-throughput fashion. Cells can optionally be assayed in multiplemulti-well devices simultaneously (for example, connected to the sameimpedance analyzer, engaged with a device station that can engage morethan one multi-well device, or connected to separate impedanceanalyzers) to increase high-throughput capacity.

Systems for monitoring cell-substrate impedance that comprise one ormore multiwell devices, an impedance analyzer, and a device station aredescribed herein and are preferred but not required for use in themethods of the present invention.

The cells used in the methods of the present invention can be any IgEresponsive cells, but are preferably mast cells isolated from differentindividuals. In addition to impedance monitoring, mast cells isolatedfrom individuals can be analyzed for genetic markers. These geneticmarkers include, as nonlimiting examples, SNPs, mutations, alternativeRNA splicing variants, gene expression profiles, and protein expressionprofiles. Methods of analyzing genetic markers using cell isolated fromindividuals is well-known in the art.

In the assay procedure, Ig E can be added before, after, or simultaneouswith the addition of the antigen. IgE can be added to wells at aconcentration of from about 10 nanograms per milliliter to about 1microgram per milliliter. Optionally, one well of the device thatcomprises an electrode array and cells does not receive IgE to provide acontrol well.

Antigens used in the assays can be antigens, allergens, or crosslinkers.The concentration added to the wells can be from about 1 nanogram permilliliter to about 1 microgram per milliliter. Antigens can be chosenfor their characteristic propensity to be identified as allergens amongmembers of a population.

The method can be used to correlate a genetic marker with theantigen-IgE response of cells, such as but not limited to mast cells, asa result of by using cell-substrate impedance technology. Impedance ispreferably monitored during two or more phases of the experiment.Depending on the experimental protocol, impedance is preferablymonitored after adding IgE to the one or wells, and is preferablymonitored before and after adding IgE to the one or wells. Impedance ispreferably monitored after the addition of test compound, before andafter the addition of IgE. Impedance can also be monitored after theaddition of test compound and IgE, before and after addition antigen. Insome preferred embodiments, impedance is monitored after the addition oftest compound and before addition of IgE to the wells, and after theaddition of IgE to the wells and before the addition of antigen,allergen, or crosslinker to the wells, and after the addition ofantigen, allergen, or crosslinker to the wells.

Impedance is preferably monitored at two or more time points.Preferably, impedance is monitored at least three time points.Preferably, impedance is monitored at least three time points at two ormore phases of the assay. Impedance at each time point can be monitoredat one or at more than one frequency. For example, impedance valuesbefore and after addition of IgE, and/or before and after addition ofantigen can be compared for each well. Impedance data can be comparedfor wells that comprise genetically manipulated cells and control wellscomprise cells that are not genetically manipulated. Impedance data canbe used to calculate a cell index, which can be used for comparisons.Such comparisons can be used to identify genetic markers that affect theresponse of cells to IgE stimulation.

Data obtained from impedance monitoring can be used to correlate geneticmarkers assigned to the cells with the responses of the cells to IgEstimulation. Identification of a genetic marker that correlates with theresponsiveness of cells to IgE can be used to develop strategies fordeveloping therapies that moderate the IgE response.

An exemplary assay is as follows:

-   -   1) Isolate mast cells from two or more individuals potent to        allergic reactions    -   2) Identify genetic markers of interest in the individuals    -   3) Incubate the mast cells (or differentiate stem cells to mast        cells in the presence of specific differentiation factors)    -   4) Transfer the cells to impedance monitoring devices and        monitor the cell growth    -   5) Add IgE (human) with or without an allergen    -   6) Quantify mast cell impedance response    -   7) Correlate genetic markers with mast cell response

Genetic analysis can be conducted using standard RFLP and SNP analysis,RNA and protein expression profiling, and RNA splicing detection methodsas they are known in the art. Preferably, assays are performed in a highthroughput manner using cells from a very large number of individuals toprovide robust correlations of genetic markers and IgE responses.

E.4 Method for Screening Discovering and Validating Chemical Structuresof Antigen (Allergen) or Half Antigen Binding the IgE Receptor Leadingto Receptor Cross-Linking

The present invention also provides methods of identifying antigens orhalf-antigens that can cause an allergic response. The method comprises:a) providing a device for cell-substrate impedance monitoring thatcomprising at least one well that comprises at least one electrodearray; b) connecting an impedance analyzer to the device; c) introducingcells into one or more wells of the device that comprise an electrodearray; d) adding IgE to the one or more wells comprising cells; e)adding at least one suspected antigen or at least one suspected allergenor half-allergen to the one or more wells comprising cells; and e)monitoring cell-substrate impedance of the one or more wells comprisingcells.

In the methods of the present invention, a device for cell-substrateimpedance monitoring is a device that comprises a substrate having oneor more electrode arrays on its surface each of which is encompassed bya fluid container in the form of a well. Each of the one or moreelectrode arrays comprises two electrodes or electrode structures, wherethe two electrodes or electrode structures have substantially the samesurface area, in which the device, when connected to an impedanceanalyzer, can detect impedance changes at one or more frequencies due tochanges in cell number, cell size, cell morphology, cell attachment tothe substrate, or the quality of cell attachment to the substrate.

The cells can be any cells whose response or possible response to IgEstimulation is of interest. Preferred cells are mast cells, eosinophils,basophils, or genetically engineered cells. In some preferredembodiments, the cells are of mammalian origin. In some preferredembodiments, the cells are mast cells isolated from individuals.

Ig E can be added before, after, or simultaneous with the addition ofthe antigen, allergen, or half-allergen. IgE can be added to wells at aconcentration of from about 10 nanograms per milliliter to about 1microgram per milliliter. Preferably, one well of the device thatcomprises an electrode array and cells does not receive IgE to provideat least one control well, but this is not a requirement of the presentinvention.

Antigens, allergens, or half-allergens can be known or suspectedantigens, allergens, or allergens. The concentration added to the wellscan be from about 1 nanogram per milliliter to about 1 microgram permilliliter. Different wells may receive different concentrations of anantigen, allergen, or half-allergens. Or, for replica purposes, multiplewells may receive same concentrations of an antigen, allergen orcrosslinker. Preferably, one well of the device that comprises anelectrode array and cells does not receive antigen, allergen, orhalf-allergen to provide at least one control well, but this is not arequirement of the present invention.

The method can be used to assess and quantify the morphological changesthat occur in mast cells as a result of IgE stimulation and IgEcross-linking with an allergen by using cell-substrate impedancetechnology. Impedance is preferably monitored during two or more phasesof the experiment. For example, in one embodiment impedance is monitoredbefore and after the addition of antigen, allergen, or half-allergen tothe wells. In another embodiment, impedance is additionally monitoredbefore and after the addition of IgE to the wells. In yet anotherembodiment, impedance can be monitored before and after the addition ofIgE plus antigen, allergen, or half-allergen to the wells. Impedance canbe also be monitored after the addition of IgE to the wells and beforethe addition of antigen, allergen, or half-allergen to the wells, aswell as after the addition of antigen, allergen, or half-allergen to thewells. In some preferred embodiments, impedance is monitored beforeaddition of IgE to the wells, after the addition of IgE to the wells andbefore the addition of antigen, allergen, or half-allergen to the wells,and after the addition of antigen, allergen, or half-allergen to thewells.

Impedance can be monitored at two or more time points. Preferably,impedance is monitored at least three time points. Preferably, impedanceis monitored at least three time points at two or more phases of theassay. Impedance at each time point can be monitored at one or at morethan one frequency. Data obtained from impedance monitoring can be usedto assess morphological changes to cells that result from IgEstimulation. For example, impedance before and after addition of IgE,and/or before and after addition of antigen, allergen, or half-allergencan be compared for each well. Impedance data can be compared for wellsthat receive IgE and control wells that do not receive IgE. Impedancedata can be compared for wells that receive antigen, allergen, orhalf-allergen and control wells that do not receive antigen, allergen,or crosslinker. Impedance data can be used to calculate a cell index,which can be used for comparisons.

One exemplary protocol is as follows:

-   -   (1) Mast cells are seeded into wells of an impedance monitoring        device.    -   (2) The cells are monitored electronically using an impedance        monitoring system for a given amount of time.    -   (3) The IgE that is specific for the allergen or the half        allergen will be added to the cells.    -   (4) Cellular response will be monitored by using the system    -   (5) After a pre-determined increment of time, the antigen or        half antigen that is specific for the IgE in step (3) will be        added.    -   (6) Cellular response will continue to be monitored using the        system.

Example 1

As an example, we describe here the use of the use of a cell-substrateimpedance monitoring system of the present invention to measure andmonitor the morphological changes that occur as a result of IgE-mediatedstimulation of RBL-2H3 cells (ATCC) in the presence or absence of anantigen. The cell-substrate impedance monitoring system has a devicestation that can engage 6 16× devices as depicted in FIG. 4. It also hasan impedance analyzer, and software that directs impedance measurementand recording and analysis of impedance data.

RBL-2H3 cells were seeded in the 16× device chamber (depicted in FIG. 2)at 20,000 cells/well and the attachment and growth of the cells in the37° C. tissue culture incubator were monitored in real-time using thecell-substrate monitoring system. After 22 hours mouse monoclonalanti-dinitrophenyl (DNP) antibody (Clone SPE-7, Sigma) was added atfinal concentration of 1 microgram/mL. As a control, a non-specificmouse IgG was added at a final concentration of 1 microgram/mL. Thedevice chambers were returned to the incubator and recording wasresumed. At 24 hours post-IgE stimulation the media in the chamber wasaspirated and 150 microliters of fresh media was added to the cells.Recording was resumed for 1 hour followed by the addition of DNP-albumin(Sigma) at a final concentration of 1 microgram/ml. The recording wascontinued for an additional 4 hours.

RBL-2H3 mast cells have been used extensively as a model system toinvestigate the signaling pathways that are initiated as a result ofIgE-mediated binding to and stimulation of the high affinityFc(epsilon)RI receptors located on the membrane. Mast cell degranulationis accompanied by distinct morphological changes which involve the actincytoskeleton (Pfeiffer et al. J Cell Biol. 1985 December;101(6):2145-55). Engagement of Fc(epsilon)RI by IgE, even in the absenceof cross-linking by an antigen, leads to dose-dependent degranulationand morphological changes (Oka et al. Am J Physiol Cell Physiol. 2003Sep. 17). This IgE-mediated dose-dependent phenomenon is also observedusing the impedance system as shown in FIG. 13. It is observed as anabrupt shift in the trace of the cell index number at 22 hours,immediately after the administration of the IgE. The duration of thesignal for the IgE concentrations that illicit a change lasts forapproximately 6 hours. Such dynamic changes are consistent withmorphological dynamics observed by microscopy and degranulation detectedby enzymatic assays. Importantly, the shift in the trace does not occurin those wells which have IgG, indicating that the response is specificfor IgE. The peak amplitude of the recording is directly dependent onthe concentration of the IgE used to stimulate the cells. The durationof the signal for the IgE concentrations that illicit a change lasts forapproximately 6 hours.

Once RBL-2H3 mast cells have been sensitized with IgE, furtherdegranulation and actin-mediated morphological dynamics can be elicitedby the administration of the antigen to which the IgE has been raisedagainst. In RBL-2H3 cells this has been observed by immunofluorescencemicroscopy as extensive membrane ruffling and lamellapodia formation onthe apical surface of the cells. The phenomenon of antigen-mediatedIgE-dependent mast cell degranulation and morphological dynamics canalso be monitored and measured using cell-impedance monitoring system.20 hours after IgE addition the media is removed and replaced by freshmedia. The change in media also induces a non-specific transient changein the signal which immediately returns to baseline after 30 minutes.Application of DNP-albumin at this point induced an abrupt shift in therecording which peaks at about 15-20 minutes and returns to baselineabout 1 hour later. The duration of the shift in the signal is much moretransient under these conditions, when compared to IgE alone stimulation(3 hours vs. 6 hours).

Example 2

The following example is provided to show how a real-time cellelectronic sensing system and methods can be used in real-timemonitoring of IgE-mediated mast cell activation. The methods and devicesof the present invention are not limited to those described in theExamples. Indeed, within the scope of the present invention, there areother specific methods and approaches to conduct assays for monitoringIgE-mediated mast cell signaling and activation.

Introduction

The work presented here describes an assay for IgE-mediated mast cellactivation using measurements of cell-substrate impedance. The method isbased on quantification in real time of the morphological, cytoskeletaland cell adhesion changes that arise as a response to multivalentantigen aggregation of the IgE-Fc(epsilon)RI complex on the surface ofmast cells. Because the electronic assay readout relies onmorphological, cytoskeletal and adhesive dynamics, which are intrinsicmast cell responses to the antigen and an essential part of the mastcell activation program, it precludes the need for establishing reportercell lines or using any other reagent or cellular manipulation. Sincethe assay is performed in real time, both antigen-dependent andindependent responses to IgE-mediated activation of mast cells can bemonitored in the same assay. The assay is readily adaptable to a 96 wellformat and as shown here can be used to assay for pharmacologicalinhibitors of mast cell activation in high throughput manner.

Materials and Methods

Cells and Reagents

The RBL-2H3 cell line was purchased from American Type CultureCollection (ATCC) and maintained in DMEM containing 10% fetal bovineserum at 37° C. and 5% CO₂. Mouse monoclonal anti-dinitrophenyl IgEantibody (Clone SPE-7) and DNP-HSA were purchased from Sigma Aldich (St.Louis, Mo.). The Src-specific inhibitor SU6656, MEK-specific inhibitorPD98059 and the PLC-specific inhibitor U73122 were purchased fromCalbiochem (La Jolla, Calif.). The PKC-specific inhibitorBisindolylmaleimide and the Syk inhibitor Piceatannol were purchasedfrom Sigma. Rhodamine phalloidin was obtained from Molecular Probes(Eugene, Oreg.). Lab-Tek chamber slides were purchased through VWRScientific.

Cell-Substrate Impedance Monitoring System

The real-time cell-substrate impedance monitoring system comprises threecomponents, an impedance analyzer, a device station, and one or more 16×microtiter devices. Microelectrode sensor arrays were fabricated onglass slides using lithographical microfabrication methods and theelectrode-containing slides were assembled to plastic trays to form 16electrode-containing wells. The device station of the system receivesthe 16× microtiter devices and is capable of electronically switchingany one of the wells to the sensor (impedance) analyzer for impedancemeasurement. In operation, the devices with cells cultured in the wellsare placed into a device station that is located inside an incubator.Electrical cables connect the device station to the sensor (impedance)analyzer. Under the impedance monitoring system's software control, theimpedance analyzer can automatically select wells to be measured andcontinuously conduct impedance measurements. The impedance data from theanalyzer is transferred to a computer, analyzed and processed byintegrated software.

Impedance measured between electrodes (electrode elements) in anindividual well depends on electrode geometry, ionic concentration inthe well and whether there are cells attached to the electrodes. In theabsence of cells, electrode impedance is mainly determined by the ionenvironment both at the electrode/solution interface and in the bulksolution. In the presence of cells, cells attached to the electrodesensor surfaces will alter the local ionic environment at theelectrode/solution interface, leading to an increase in the impedance.The more cells there are on the electrodes, the larger the increase incell-electrode impedance. Furthermore, the impedance change also dependson cell morphology and the extent to which cells attach to theelectrodes.

To quantify cell status based on the measured cell-electrode impedance,a parameter termed Cell Index is derived, according to

${CI} = {\max\limits_{{i = 1},\ldots\mspace{11mu},N}\left( {\frac{R_{cell}\left( f_{i} \right)}{R_{b}\left( f_{i} \right)} - 1} \right)}$where R_(b)(f) and R_(cell)(f) are the frequency dependent electroderesistances (a component of impedance) without cells or with cellpresent, respectively. N is the number of the frequency points at whichthe impedance is measured. Thus, Cell Index is a quantitative measure ofthe status of the cells in an electrode-containing well. Under the samephysiological conditions, more cells attached on to the electrodes leadsto larger R_(cell)(f) value, leading to a larger value for Cell Index.Furthermore, for the same number of cells present in the well, a changein the cell status such as morphology will lead to a change in the CellIndex. For example, an increase in cell adhesion or cell spreading leadsto larger cell-electrode contact area which will lead to an increase inR_(cell)(f) and thus a larger value for Cell Index.Fluorescence Microscopy

RBL-2H3 cells were seeded in 16 well Lab-Tec chamber slides and allowedto attach and spread for 6 hours. The cells were stimulated withanti-DNP IgE at a final concentration of 100 ng/mL or a non-specificmouse IgG at 100 ng/mL and then 16 hours later, the media was aspirated,replaced with fresh media and treated with 100 ng/mL DNP-BSA for theindicated time and then fixed with 4% parafarmaldehyde. The cells werewashed 3× with PBS, permeablized in PBS containing 0.2% TX-100 andblocked in PBS containing 0.5% BSA. The cells were then stained withrhodamine-phalloidin for 30 minutes, washed 3× with PBS and visualizedand imaged using the tritc filter on a Nikon E400 epi-fluorescencemicroscope and Nikon ACT software.

2.5. Beta-Hexosaminidase Assay

RBL-2H3 cells growing in 96 well plates were washed and incubated inTyrode buffer (10 mM Hepes, pH 7.4, 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2,1 mM MgCl2, 5.6 mM Glucose, and 0.1% BSA) and stimulated with 100 ng/mLanti-DNP IgE. After 2 hours the supernatant was removed and the cellmonolayer was lysed in Tyrode buffer containing 0.5% TX-100.Hexosaminidase activity was measured in both supernatant and the cellmonolayer using the substrate4-nitrphenyl-2acetamido-2-deoxy-b-D-glucopyranoside (1 mg/mL). After 1hour incubation at 37° C., the reaction was stopped by the addition of 2volumes of 0.4 M glycine pH 10.7. The absorbance at 405 nm was read inMolecular Devices ELISA reader.

Impedance Assay

20,000 RBL-2H3 cells were seeded per well of a 16× microtiter device andmonitored by the impedance measuring system. The cells were allowed toattach and spread for 5-24 hours prior to the addition of IgE at theindicated final concentration. The cell-electrode impedance wascontinuously measured and the corresponding, time dependent Cell-Indexvalues were derived and recorded.

Impedance Monitoring of RBL-2H3 Mast Cell Activation

IgE-mediated RBL-2H3 mast cell activation in the presence of antigenleads to initiation of signaling cascade resulting in degranulation ofsecretory vesicles which contain mediators of allergic reaction such ashistamine. In addition, IgE-mediated stimulation through Fc(epsilon)RIalso leads to dramatic remodeling of the actin cytoskeleton (Oliver etal., 1997). Since monitoring of cell-electrode impedance providesinformation about the parameters of cell morphology and adhesion wesought to determine the impedance response of RBL-2H3 mast cells thatwere pre-sensitized with IgE in the presence of antigen application.

RBL-2H3 mast cells were seeded onto the surface of 16× microtiter platedevices having integrated microelectronic sensor arrays in the bottom ofeach well. The cells were allowed to adhere to the surface of thesensors and 18 hours later were sensitized with anti-DNP IgE (FIG. 14).Approximately 24 hours later DNP-BSA at a final concentration of 100ng/mL was applied to the cells to induce oligomerization of theIgE-bound Fc(epsilon)RI receptor and induce mast cell activation. Thecell-electrode impedance measurements were continuously monitored usingthe impedance monitoring system. As shown in FIG. 14, DNP-BSAapplication induced an immediate and transient increase in the impedancevalue which was detectable within 5 minutes of DNP-BSA application,maximal by 30 minutes and returned to baseline in approximately 2.5hours. IgE-mediated activation of mast cells not only led to dramaticmorphological changes (Pfeiffer et al., 1985) but also to augmentationof integrin-mediated cell adhesion (Wyczolkowska et al., 1994), both ofwhich contributed to cell-electrode impedance measurements using theimpedance monitoring system.

Impedance Measurement of Mast Cell Activation Correlates withCytoskeletal Dynamics and Degranulation

To determine if the IgE-mediated cell-electrode impedance increasecorrelates with RBL-2H3 mast cell activation, both IgE-mediatedmorphological dynamics and mediator release were monitored.

RBL-2H3 mast cells were sensitized and activated as described above andat the indicated time points, fixed with parafarmaldehyde and stainedwith rhodamine-phalloidin to visualize the actin cytoskeleton (FIG.15A). As seen in FIG. 15A and shown previously (Pfeiffer et al., 1985;O'Luanaigh et al., 2002; Powner et al., 2002), DNP-BSA-mediatedcross-linking of the Fc(epsilon)RI receptor leads to time-dependentremodeling of the actin cytoskeleton. The cells undergo extensiveruffling which is apparent as early as 2.5 minutes post IgE stimulation,followed by morphological changes which lead to cell spreading andformation of lamellapodia. The peak cytoskeletal reorganization isobserved at 30-45 minutes post-IgE stimulation which correlates directlywith the peak cell-electrode impedance response using the impedancemonitoring system. As a control, RBL-2H3 mast cells were also sensitizedwith an irrelevant IgG and subsequently cross-linked with DNP-BSA. Noobvious cytoskeletal and morphological changes were observed.

As an additional marker for RBL-2H3 mast cell activation,beta-hexosaminidase activity was also measured in response to IgEstimulation in the presence of antigen cross-linking. The enzymebeta-hexosaminidase is stored within the secretory vesicles and is amarker for mast cell degranulation. It has been shown thatbeta-hexosaminidase is released into the culture media in response toantigen-mediated cross-linking of IgE-bound Fc(epsilon)RI on the surfaceof mast cells (Razin et al., 1983). RBL-2H3 cells were sensitized withanti-DNP IgE, activated by application of DNP-BSA andbeta-hexoseaminidase activity was measured as described in materials andmethods section. Antigen cross-linking leads to a two and a half to 4fold increase in beta-hexosaminidase depending on the experiment (FIG.15B). Taken together, IgE-mediated mast cell-electrode impedanceincrease correlates directly with morphological changes anddegranulation which is characteristic of mast cell activation.Therefore, cell-electrode impedance measurements can be used as readoutfor mast cell activation.

Cell-Substrate Monitoring of RBL-2H3 Sensitization Step

It has recently been shown that high concentrations of monomeric IgEinduce mast cell activation in the absence of antigen cross-linking (Okaet al., 2004). Accordingly, we wanted to determine if highconcentrations of IgE alone may induce an increase in cell-electrodeimpedance response. RBL-2H3 mast cells growing on the surface of 16×microtiter cell-substrate impedance monitoring devices were stimulatedwith increasing amounts of IgE-ranging from 15 nanogram/mL to 1micrograms/mL and their impedance response was continuously monitored(FIG. 16A). IgE application alone leads to a dose-dependent increase incell-electrode impedance response which correlated with morphologicaldynamics and mediator release. To determine if the initial concentrationof IgE used to sensitize the cells influences the subsequentantigen-mediated response, RBL-2H3 cells were sensitized with theindicated concentrations of anti-DNP IgE and after 16 hours wereincubated with 100 nanograms/mL final concentration of DNP-BSA. As shownin FIG. 16B, the DNP-BSA-mediated response is inversely proportional tothe initial anti-DNP IgE concentration used to sensitize the cells.Sensitizing with 1 microgram/ml anti-DNP IgE leads to negligibleincrease in cell-electrode impedance response when cross-linked with themulti-valent antigen DNP-BSA while sensitization with 15 nanograms/mLanti-DNP IgE resulted in a robust increase in cell-electrode(cell-substrate) impedance in the presence of DNP-BSA aggregation.Furthermore, if the increment of time between High IgE application andDNP-BSA addition is increased to 24 hours, then the cell will undergoantigen-mediated activation.

In conclusion these experiments illustrate that IgE concentration andthe timing between IgE sensitization and subsequent antigen stimulationis critical and should be taken into account. Additionally, theseexperiments further illustrate the advantage of real-time monitoring ofmast cell activation on microelectronic cell-substrate impedance sensorarrays with respect to cell status and response at any given time point.

Pharmacological Inhibition of Mast Cell Activation as Detected by theCell-Substrate Impedance Monitoring System

Antigen-mediated aggregation of the IgE-bound Fc(epsilon)RI triggers asignaling cascade which involves a number of signaling proteins such asprotein kinases, protein phosphatases, and phospholipases amongst otherswhose activity and function is indispensable for mast cell activation(Turner and Kinet, 1999). Accordingly, we were interested in determiningif pharmacological inhibitory effects of some of these signalingproteins can be monitored by mast cell-electrode impedance measurement.

RBL-2H3 cells were seeded in a 16× microtiter plate device andpre-stimulated with anti-DNP-IgE were incubated for 1 hour with theindicated doses of the PKC-specific inhibitor Bisindolylmaleimide andsubsequently treated with 100 ng/mL DNP-BSA (FIG. 17). Thedose-dependent inhibition of RBL-2H3 mast cell activation byBisindolylmaleimide can be monitored on the cell-substrate monitoringsystem in real-time. A number of other pharmacological agents such asSU6656, specific for Src family kinases, U73122, specific forphospholipase C, Piceatannol, specific for Syk tyrosine kinase andPD98059 specific for MEK were also tested. The IC-50 value for thesepharmacological inhibitors at peak response was calculated and is shownin Table I. All the inhibitors with the exception of PD 98059 dosedependently inhibited antigen-mediated RBL-2H3 mast cell activation. ThePLC-specific inhibitor U73122 displayed potent inhibitory effect with anIC-50 value of around 1 microM. The Src and Syk specific inhibitors alsoinhibited the antigen-mediated response although at higherconcentrations (Table I). The MEK-specific inhibitor PD 98059 hadminimal effect on IgE-mediated RBL-2H3 response at the concentrationstested.

In order to ascertain that inhibitor-mediated abrogation of thecell-electrode impedance response correlates with inhibition of mastcell activation, the beta-hexosaminidase activity of IgE stimulatedRBL-2H3 cells were measured in the presence of pharmacological agents.According to FIG. 18, SU6656, U73122, Bisindolylmaleimide andPiceatannol abrogated the anti-DNP IgE mediated stimulation ofbeta-hexosaminidase activity in the presence of antigen aggregationwhereas the MEK specific inhibitor had minimal effect. These findingsare in agreement with previously published data indicating that the Src,PLC, PKC and Syk specific inhibitors completely block IgE-mediated mastcell degranulation and activation (Oliver et al., 1994; Amoui et al.,1997; Moriya et al., 1997; Tedeschi et al., 2000). In summary, theresults presented here indicate that real-time monitoring ofIgE-mediated mast cell activation on microelectronic cell sensor arraysoffer a convenient way of assessing mast cell activation. The assay doesnot require any cellular manipulation such as labeling, fixation orlysis. The assay was validated by demonstrating that the cell-electrode(cell-substrate) impedance measurement correlates directly withIgE-mediated mast cell activation as measured by actin cytoskeletondynamics and mediator release. Furthermore, previously characterizedspecific inhibitors of mast cell activation pathway inhibit mast cellactivation as measured by the cell-substrate impedance monitoringsystem, further validating this assay.

TABLE I IC-50 determination of pharmacological agents inhibiting mastcell activation monitored by cell-substate impedance monitoring.Compound Target IC50 ± SD (μM) Bisindolylmaleimide Protein Kinase C 2.9± 1.7 (N = 3) SU6656 SRC 5.8 ± 3.5 (N = 5) U73122 Phospholipase C 0.93 ±0.6 (N = 5) Piceatannol Syk 1 (N = 1)Discussion

Cell-electrode impedance reading is primarily influenced by three mainparameters: the number of cells seeded on microelectronic sensor arrays,the shape of the cell and the strength of cell adhesion to the electrodesurface. IgE-mediated RBL-2H3 mast cell activation is accompanied by anincrease in effective surface area due to fusion of secretory granules,dramatic actin cytoskeleton rearrangement as well as an increase inintegrin-mediated adhesion (Pfeiffer et al., 1985; Wyczolkowska et al.,1994). Morphological dynamics combined with an increase in adhesiveinteraction of the cell with the electrode surface leads to an increasein antigen-dependent IgE-mediated mast cell electrode impedance value.Furthermore, pharmacological inhibitors of signaling proteins whichparticipate in IgE-mediated mast cell activation inhibit IgE-mediatedmast cell activation in a dose-dependent manner (FIG. 17 and Table I).

According to the current paradigm for mast cell activation,antigen-mediated cross-linking of the IgE-bound Fc□RI leads tophosphorylation of critical tyrosines in the immunoreceptortyrosine-based activation motifs (ITAM) by Src family kinase, Lyn(Turner and Kinet, 1999). Phosphorylation of the ITAMS serves as adocking site for the Syk protein tyrosine kinase, which subsequentlyleads to its activation and phosphorylation of its downstream substratessuch as phospholipase C gamma (PLCgamma). PLCgamma catalyzes thehydrolysis of membrane phospholipids to generate inositol1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). IP₃ mobilizescalcium from internal reserves in the endoplasmic reticulum and bothcalcium and DAG lead to general activation of protein kinase C (PKC)family as well as other kinases and signaling proteins. Activation ofIgE-mediated kinase pathways ultimately results in mast celldegranulation and release of inflammatory mediators. Accordinglypharmacological inhibitors of Src, Syk, PLCgamma and PKC led to adose-dependent inhibition of IgE-mediated RBL-2H3 mast activation whichcan be monitored by the cell-electrode impedance measurement. Thecell-electrode impedance values of the mast cells treated with thesepharmacological inhibitors correlated with mast cell degranulation asmeasured by beta-hexosaminidase activity (FIG. 18). Utilization of thesepharmacological inhibitors further validates this assay and indicatesthat the cell-substrate impedance monitoring system can be used toassess mast cell activation.

Several lines of recent evidence indicate that IgE-alone mediatedinteraction with mast cells has crucial biological functions thatsurpass its role as merely a sensitizer of mast cells (Kawakami andGalli, 2002). Two different groups have shown that application ofmonomeric IgE without antigen-mediated aggregation inducesphosphorylation and activation of mitogen activated protein kinases(MAPKs) and AKT leading to enhanced mast cell survival (Asai et al.,2001; Kalesnikoff et al., 2001; Kitaura et al., 2003). However, the samepapers also showed that monomeric IgE did not induce mast celldegranulation. In contrast, another recently published report indicatesthat high concentrations of monomeric IgE can induce mast cellactivation in itself without the need for antigen-mediated ligation (Okaet al., 2004). According to the authors, IgE alone-mediated mast cellactivation as measured by mediator release and actin cytoskeletonrearrangement was indistinguishable from antigen-mediated cross-linking(Oka et al., 2004). According to our results application of highconcentrations of monomeric IgE alone also elicited an increase in mastcell-electrode impedance value, indicating mast cell activation (FIG.16). However, the duration of the response was prolonged when comparedto the antigen-mediated mast cell activation. While the molecularmechanism of antigen-independent and antigen-dependent mast cellactivation response remains to be clarified, it is clear that monomericIgE can illicit biologically important responses. We've also determinedthat the initial IgE concentration used to sensitize the cellsultimately determines the amplitude and duration of the subsequentantigen-mediated mast cell activation. Sensitization with highconcentrations of IgE (1 microgram/mL) resulted in very lowantigen-dependent mast cell response, while sensitization with lowconcentrations of IgE (15 nanogram/mL) resulted in mast cell responsewith much higher duration and amplitude (FIG. 16B). The lack of asignificant antigen-mediated mast cell response with high concentrationof IgE-sensitized mast cells more likely is due to the fact that mastcells have not had an opportunity to fully reform their secretorygranules after IgE-mediated activation and this is supported by the factthat increasing the time increment between sensitization with 1microgram/mL IgE and the cross-linking step will ultimately result inantigen-mediated mast cell activation.

In summary, the real-time and label-free IgE mediated mast cellactivation assay described here should provide researchers inpharmaceutical industry as well as academia with a new and convenienttool to assess and quantify IgE-mediated mast cell activation. Theadaptability of this assay to 96× microtiter plates with microelectronicsensors integrated in the bottom of the wells makes it ideal for highthroughput analysis to screen large chemical compound libraries.Furthermore, microelectronic cell sensor technology in general can beused to assess other receptor ligand interactions.

All of the references cited herein, including patents, patentapplications, and publications, and including references cited in theBibliography, are incorporated by reference in their entireties.

Headings are for the convenience of the reader and do not limit thescope of the invention.

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1. A method of performing a cell-based assay that monitorscell-substrate impedance in response to one or more test compounds,comprising: (a) providing a cell-substrate impedance monitoring system,comprising: (i) at least one multiple-well cell-substrate impedancemeasuring device, wherein at least two of the multiple wells comprise anelectrode array at the bottom of the well, wherein each electrode arraycomprises two electrode structures and each electrode structurecomprises multiple electrode elements, further wherein each electrodearray is individually addressed; (ii) an impedance analyzer; (iii) adevice station comprising electronic circuitry capable of engaging saiddevice and selecting and connecting electrode arrays within any of themultiple wells to the impedance analyzer; (iv) a software programcapable of controlling the device station and performing dataacquisition and data analysis from said impedance analyzer; (b)introducing cells into one or more wells of said system that compriseelectrode arrays; (c) adding at least one test compound to said one ormore wells; (d) monitoring cell-substrate impedance of said one or morewells before and after adding said at least one test compound; and (e)analyzing impedance values and calculating cell index values before andafter adding said at least one test compound and providing informationabout cell responses to said at least one test compound.
 2. The methodaccording to claim 1, wherein impedance is monitored at three or moretime points.
 3. The method according to claim 1, further comprisingintroducing cells to at least one well to which the test compound is notadded to create a control well.
 4. The method according to claim 1,wherein said cell index values are compared.
 5. The method according toclaim 1, wherein said cell index values are used as an indicator ofcytotoxicity.
 6. The method according to claim 1, wherein said analyzingfurther comprises providing information selected from the groupconsisting of cell proliferation, cell death, cell adhesion, cellapoptosis, cell differentiation, cell survival, cytotoxicity, cellmorphology, cell quantity, cell quality, time-dependent cytotoxicity ofa compound, IgE-mediated cell activation or stimulation, receptor-ligandbinding, viral and bacterial toxin mediated cell pathologic changes,presence and quantity of neutralizing antibodies, specific T-cellmediated cytotoxic effects, presence and affinity of ligand-receptorbinding.
 7. The method of claim 1, wherein said analyzing furthercomprises providing information selected from the group consisting ofcell attachment or adhesion status on the substrate including on theelectrodes, the degree of cell spreading, the attachment area of a cell,the degree of tightness of cell attachment, cell morphology, cell growthor proliferation status, number of viable cells and/or dead cells in thewell, cytoskeleton change and re-organization and number of cells goingthrough apoptosis and/or necrosis.
 8. The method of claim 7, whereinsaid analyzing provides information about cell morphology, wherein saidtest compound binds to a receptor on the cell surface and said bindingleads to a change in cell morphology.
 9. The method according to claim1, wherein cells are added to at least two wells of said device, each ofwhich comprises an electrode array, and wherein impedance is monitoredfrom at least two wells that comprise cells and an electrode array. 10.The method according to claim 1, wherein said monitoring impedancecomprises monitoring impedances at three or more time points spaced atregular intervals.
 11. The method according to claim 1, wherein saidcells are primary cells isolated from a species or cells obtained fromcell lines.
 12. The method according to claim 1, wherein impedance ismonitored at one frequency.
 13. The method according to claim 1, whereinimpedance is monitored at multiple frequencies.
 14. The method accordingto claim 13, wherein impedance is monitored at multiple frequencies at atime point.